Antibodies that immunospecifically bind to b lymphocyte stimulator protein

ABSTRACT

The present invention relates to antibodies and related molecules that immunospecifically bind to B Lymphocyte Stimulator. The present invention also relates to methods and compositions for detecting or diagnosing a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or inappropriate function of B Lymphocyte Stimulator comprising antibodies or fragments or variants thereof or related molecules that immunospecifically bind to B Lymphocyte Stimulator. The present invention further relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or inappropriate B Lymphocyte Stimulator function comprising administering to an animal an effective amount of one or more antibodies or fragments or variants thereof or related molecules that immunospecifically bind to B Lymphocyte Stimulator.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending U.S. patent application Ser. No. 12/605,202, filed Oct. 23, 2009, which is continuation of copending U.S. patent application Ser. No. 11/054,515, filed Feb. 10, 2005, which is a continuation in-part of U.S. patent application Ser. No. 10/293,418, filed Nov. 14, 2002, which issued as U.S. Pat. No. 7,220,840, and U.S. patent application Ser. No. 09/880,748, filed Jun. 15, 2001, which issued as U.S. Pat. No. 7,138,501. U.S. patent application Ser. No. 11/054,515 claims the benefit of U.S. Provisional Application No. 60/543,296, filed Feb. 11, 2004, and U.S. Provisional Application No. 60/580,347, filed Jun. 18, 2004. U.S. patent application Ser. No. 10/293,418 claims the benefit of U.S. Provisional Application No. 60/331,469, filed Nov. 16, 2001, and U.S. Provisional Application No. 60/340,817, filed Dec. 19, 2001. U.S. patent application Ser. No. 10/293,418 is a continuation-in-part of U.S. patent application Ser. No. 09/880,748, filed Jun. 15, 2001, which issued as U.S. Pat. No. 7,138,501. U.S. patent application Ser. No. 09/880,748 claims the benefit of U.S. Provisional Application No. 60/212,210, filed Jun. 16, 2000, U.S. Provisional Application No. 60/240,816, filed Oct. 17, 2000, U.S. Provisional Application No. 60/276,248, filed Mar. 16, 2001, U.S. Provisional Application No. 60/277,379, filed Mar. 21, 2001, and U.S. Provisional Application No. 60/293,499, filed May 25, 2001. Each of the applications identified above is herein incorporated by reference in their entireties.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 4,949,952 Byte ASCII (Text) file named “SequenceListing.TXT,” created on Aug. 12, 2011.

INTRODUCTION

The present invention relates to antibodies and related molecules that immunospecifically bind to B Lymphocyte Stimulator (BLyS™) protein. The present invention also relates to methods and compositions for detecting, diagnosing, or prognosing a disease or disorder associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate function of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor, comprising antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to B Lymphocyte Stimulator. The present invention further relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate B Lymphocyte Stimulator function or B Lymphocyte Stimulator receptor function, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to B Lymphocyte Stimulator.

BACKGROUND OF THE INVENTION

B Lymphocyte Stimulator (BLyS™) protein is a member of the tumor necrosis factor (“TNF”) superfamily that induces both in vivo and in vitro B cell proliferation and differentiation (Moore et al., Science 285: 260-263 (1999)). B Lymphocyte Stimulator is distinguishable from other B cell growth and differentiation factors such as IL-2, IL-4, IL-5, IL-6, IL-7, IL-13, IL-15, CD40L, or CD27L (CD70) by its monocyte-specific gene and protein expression pattern and its specific receptor distribution and biological activity on B lymphocytes. B Lymphocyte Stimulator expression is not detected on natural killer (“NK”) cells, T cells or B cells, but is restricted to cells of myeloid origin. B Lymphocyte Stimulator expression on resting monocytes is upregulated by interferon-gamma (IFN-gamma). The gene encoding B Lymphocyte Stimulator has been mapped to chromosome 13q34.

B Lymphocyte Stimulator is expressed as a 285 amino acid type II membrane-bound polypeptide and a soluble 152 amino acid polypeptide (Moore et al., 1999 supra). The membrane-bound form of B Lymphocyte Stimulator has a predicted transmembrane spanning domain between amino acid residues 47 and 73. The NH₂-terminus of the soluble form of B Lymphocyte Stimulator begins at Ala¹³⁴ of the membrane-bound form of B Lymphocyte Stimulator. Soluble recombinant B Lymphocyte Stimulator has been shown to induce in vitro proliferation of murine splenic B cells and to bind to a cell-surface receptor on these cells (Moore et al., 1999 supra). Soluble B Lymphocyte Stimulator administration to mice has been shown to result in an increase in the proportion of CD45R^(dull), Ly6D^(bright) (also known as ThB) B cells and an increase in serum IgM and IgA levels (Moore et al., 1999 supra). Thus, B Lymphocyte Stimulator displays a B cell tropism in both its receptor distribution and biological activity.

Levels of B Lymphocyte Stimulator protein have been found to be elevated in patients with autoimmune disease, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren's syndrome (Zhang et al., The Journal of Immunology, (2001) 166:6-10; Cheema et al., Arthritis and Rheumatism (2001) 44:1313-1319; and Groom et al., Journal of Clinical Investigation (2002) 109:59-68). Furthermore, administration of a soluble form of a B Lymphocyte Stimulator receptor, TACI, has been shown to alleviate the autoimmune phenotype of NZBWF1 and MRL-lpr/lpr mice (Gross et al., Nature, (2000) 404:995-999). Thus, antibodies and related molecules that immunospecifically bind to B Lymphocyte Stimulator may find medical utility in, for example, the treatment of B cell disorders associated with autoimmunity. In other embodiments, antibodies and related molecules that immunospecifically bind to B Lymphocyte Stimulator may find medical utility in for example, neoplasia or immunodeficiency syndromes.

SUMMARY OF THE INVENTION

The present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator. In particular, the invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment of human B Lymphocyte Stimulator (SEQ ID NOS:3228 and/or 3229) or B Lymphocyte Stimulator expressed on human monocytes; murine B Lymphocyte Stimulator (SEQ ID NOS:3230 and/or 3231) or B Lymphocyte Stimulator expressed on murine monocytes; rat B Lymphocyte Stimulator (either the soluble forms as given in SEQ ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey B Lymphocyte Stimulator (e.g., the monkey B Lymphocyte Stimulator polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey B Lymphocyte Stimulator, or B Lymphocyte Stimulator expressed on monkey monocytes), preferably human B Lymphocyte Stimulator. The present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate function of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor in an animal, preferably a mammal, and most preferably a human, comprising, or alternatively consisting of, use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator. Diseases and disorders which can be detected, diagnosed, or prognosed with the antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma). The present invention further encompasses methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate function of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor in an animal, preferably a mammal, and most preferably a human, comprising, or alternatively consisting of, administering to said animal an effective amount of one or more antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator. Diseases and disorders which can be prevented, treated or ameliorated by administering an effective amount of an antibody of the invention include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).

Using phage display technology, the present inventors have identified single chain antibody molecules (“scFvs”) that immunospecifically bind to B Lymphocyte Stimulator, including scFvs that immunospecifically bind to soluble B Lymphocyte Stimulator, scFvs that immunospecifically bind the membrane-bound form of B Lymphocyte Stimulator, and scFvs that immunospecifically bind to both the soluble form and the membrane-bound form of B Lymphocyte Stimulator. Antibodies of the present invention are defined as able to bind the membrane bound and/or soluble forms of B Lymphocyte Stimulator according to the assays described in Examples 1 through 19. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.

In particular, the invention relates to scFvs comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-2128, preferably SEQ ID NOS:834-872, 1570-1595, and 1886-1908, and most preferably SEQ ID NOS:1-46, 321-329, 1563-1569, and 1881-1885, as referred to in Table 1 below. In specific embodiments, the present invention relates to scFvs that immunospecifically bind the soluble form of B Lymphocyte Stimulator, said scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1563-1880, preferably SEQ ID NOS:1570-1595, and most preferably SEQ ID NOS: 1563-1569, as referred to in Table 1, below. In other embodiments, the present invention also relates to scFvs that immunospecifically bind the membrane-bound form of B Lymphocyte Stimulator, said scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1881-2128, preferably SEQ ID NOS:1886-1908, and most preferably SEQ ID NOS: 1881-1885, as referred to in Table 1 below. The present invention further relates to scFvs that immunospecifically bind both the membrane-bound form and soluble form of B Lymphocyte Stimulator, said scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1-1562, preferably SEQ ID NOS: 834-872, and most preferably SEQ ID NOS: 1-46, and 321-329, as referred to in Table 1 below. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.

The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator, said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one of the variable heavy (“VH”) domains referred to in Table 1, below, or any one of the variable light (“VL”) domains referred to in Table 1. In a preferred embodiment, antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908, as referred to in Table 1 below. In another preferred embodiment, antibodies (including molecules comprising or alternatively consisting of, antibody fragments or variants thereof) of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL domain contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908, as referred to in Table 1 below. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

The present invention also provides antibodies (including molecules comprising or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator, said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1, below, and any one of the VL domains referred to in Table 1. In a preferred embodiment, the antibodies of the invention comprise or alternatively consist of, a polypeptide having the amino acid sequence of a VH and VL domain contained in the same scFv referred to in Table 1. In another preferred embodiment, antibodies of the present invention, comprise, or alternatively consist of, a VH domain from an scFv of SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908, as disclosed in Table 1, and a VL domain from an scFv SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908, as disclosed in Table 1. In another preferred embodiment, antibodies of the present invention comprise, or alternatively consist of, the VH and VL domain from a single scFv of SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908, as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

The present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator, said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one, two, three or more of the VH complementarity determining regions (“CDRs”) (i.e., VH CDR1, VH CDR2, or VH CDR3) referred to in Table 1 and/or any one, two, three or more of the VL CDRs (i.e., VL CDR1, VL CDR2, or VL CDR3) referred to in Table 1. In one embodiment, antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDR1s referred to in Table 1 and/or any one of the VL CDR1s referred to in Table 1. In another embodiment, antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDR2s referred to in Table 1 and/or any one of the VL CDR2s referred to in Table 1. In a preferred embodiment, antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDR3s referred to in Table 1 and/or any one of the VL CDR3s referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

In another embodiment, antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator, and comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDR1s referred to in Table 1, any one of the VH CDR2s referred to in Table 1, and/or any one of the VH CDR3s referred to in Table 1. In another embodiment, antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VL CDR1s referred to in Table 1, any one of the VL CDR2s referred to in Table 1, and/or any one of the VL CDR3s referred to in Table 1. In a preferred embodiment, antibodies of the present invention comprise, or alternatively consist of, at least one, two, three, four, five, six, or more CDRs that correspond to the same scFv referred to in Table 1, more preferably where CDR1, CDR2, and CDR3 of the VL domain correspond to the same scFv or where CDR1, CDR2, and CDR3 of the VH domain correspond to the same scFv, and most preferably where all six CDRs correspond to the same scFv referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

The present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to the soluble form of B Lymphocyte Stimulator (e.g., a polypeptide consisting of amino acids 134-285 of SEQ ID NO:3228); that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator (e.g., a polypeptide consisting of amino acids 1-285 of SEQ ID NO:3228 or a B Lymphocyte Stimulator polypeptide expressed on the surface of monocytes) and/or that immunospecifically bind to both the soluble form and membrane-bound form of B Lymphocyte Stimulator. In a preferred embodiment, antibodies of the present invention immunospecifically bind to the soluble form of B Lymphocyte Stimulator and comprise, or alternatively consist of, a VH domain, VH CDR1, VH CDR2, VH CDR3, VL domain, VL CDR1, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that immunospecifically bind to the soluble form of B Lymphocyte Stimulator. In another preferred embodiment, antibodies of the present invention immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator and comprise, or alternatively consist of, a VH domain, VH CDR1, VH CDR2, VH CDR3, VL domain, VL CDR1, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator. In yet another preferred embodiment, antibodies of the present invention immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator and comprise, or alternatively consist of, a VH domain, VH CDR1, VH CDR2, VH CDR3, VL domain, VL CDR1, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that immunospecifically binds to the soluble form and membrane-bound form of B Lymphocyte Stimulator. In another preferred embodiment, antibodies of the present invention comprise, or alternatively consist of, a VH domain and a VL domain corresponding to the same scFv disclosed in Table 1, which antibodies immunospecifically bind to the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, or both the soluble form and membrane-bound form of B Lymphocyte Stimulator. Nucleic acid molecules encoding these antibodies are also encompassed by the invention. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

The present invention also provides antibodies (including molecules comprising or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to both B Lymphocyte Stimulator and APRIL (preferably to the soluble forms of each of these molecules), said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1, below, and any one of the VL domains referred to in Table 1. In a preferred embodiment, the antibodies of the invention comprise or alternatively consist of, a polypeptide having the amino acid sequence of a VH and VL domain contained in the same scFv referred to in Table 1. In another preferred embodiment, antibodies of the present invention that immunospecifically bind to both B Lymphocyte Stimulator and APRIL, comprise, or alternatively consist of, a VH domain from an scFv of SEQ ID NOS:3240-3247 as disclosed in Table 1, and a VL domain from an scFv SEQ ID NOS:3240-3247, as disclosed in Table 1. In another preferred embodiment, antibodies of the present invention that immunospecifically bind to both B Lymphocyte Stimulator and APRIL comprise, or alternatively consist of, the VH and VL domain from a single scFv of SEQ ID NOS: SEQ ID NOS:3240-3247, as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind both B Lymphocyte Stimulator and APRIL, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

The present invention also provides antibodies (including molecules comprising or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a heterotrimeric protein comprising at least one B Lymphocyte Stimulator polypeptide (preferably amino acids 134-285 of SEQ ID NO:3228), said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1, below, and any one of the VL domains referred to in Table 1. In a preferred embodiment, the antibodies of the invention that immunospecifically bind heterotrimeric protein comprising at least one B Lymphocyte Stimulator polypeptide, comprise or alternatively consist of, a polypeptide having the amino acid sequence of a VH and VL domain contained in the same scFv referred to in Table 1. In another preferred embodiment, antibodies of the present invention that immunospecifically bind heterotrimeric protein comprising at least one B Lymphocyte Stimulator polypeptide, comprise, or alternatively consist of, a VH domain from an scFv of SEQ ID NOS:3240-3247 as disclosed in Table 1, and a VL domain from an scFv SEQ ID NOS:3240-3247, as disclosed in Table 1. In another preferred embodiment, antibodies of the present invention that immunospecifically bind heterotrimeric protein comprising at least one B Lymphocyte Stimulator polypeptide, comprise, or alternatively consist of, the VH and VL domain from a single scFv of SEQ ID NOS:3240-3247, as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind a heterotrimeric protein comprising at least one B Lymphocyte Stimulator polypeptide, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

A VH domain of an amino acid sequence disclosed herein may be combined with a VL domain of an amino acid sequence disclosed herein, or other VL domains, to provide a VH/VL pairing representing an antigen-binding site of an antibody. Similarly, a VL domain of an amino acid sequence disclosed herein may be combined with a VH domain of an amino acid sequence disclosed herein, or other VH domains. Further, one or more CDRs disclosed herein may be taken from a VH or VL domain and incorporated into a suitable framework as discussed infra.

The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof (including derivatives)) comprising, or alternatively consisting of, of VH domains, VL domains and/or CDRs described herein, which antibodies, immunospecifically bind to B Lymphocyte Stimulator (e.g., soluble B Lymphocyte Stimulator and membrane-bound B Lymphocyte Stimulator) and can be routinely assayed for immunospecific binding to B Lymphocyte Stimulator using methods known in the art, such as, for example, the immunoassays disclosed infra. Antibodies and antibody fragments or variants (including derivatives) of the invention may include, for example, one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue). These alterations may be made in one or more framework regions and/or one or more CDR's. The antibodies of the invention (including antibody fragments, and variants and derivative thereof) can be routinely made by methods known in the art. Molecules comprising, or alternatively consisting of, fragments or variants of any of the VH domains, VH CDRs, VL domains, and VL CDRs whose sequences are specifically disclosed herein may be employed in accordance with the present invention. Nucleic acid molecules encoding these antibodies and molecules (including fragments, variants, and derivatives) are also encompassed by the invention.

The present invention also provides panels of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab′)₂ fragments, Fd fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies, and scFvs). The present invention further provides mixtures of antibodies, wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab′)₂ fragments, Fd fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies, and scFvs)). The present invention also provides for compositions comprising, or alternatively consisting of, one, two, three, four, five, ten, fifteen, twenty, or more antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). A composition of the invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of one or more antibodies or fragments or variants thereof. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the invention.

The present invention also provides for fusion proteins comprising an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention, and a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain). Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. A composition of the present invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.

The present invention also provides for a nucleic acid molecule, generally isolated, encoding an antibody (including molecules such as scFvs, which comprise, or alternatively consist of, an antibody fragment or variant thereof) of the invention. The present invention also provides a host cell transformed with a nucleic acid molecule of the invention and progeny thereof. The present invention also provides a method for the production of an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention. The present invention further provides a method of expressing an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention from a nucleic acid molecule. These and other aspects of the invention are described in further detail below.

The present invention also encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor function in an animal, preferably a mammal, and most preferably a human, comprising using antibodies (including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator. Diseases and disorders which can be detected, diagnosed or prognosed with the antibodies of the invention include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).

In specific embodiments, the present invention encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some immunodeficiencies). In other specific embodiments, the present invention encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency).

The present invention further encompasses methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor function in an animal, preferably a mammal, and most preferably a human, comprising administering to said animal an effective amount of one or more antibodies (including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator. Diseases and disorders which can be prevented, treated or inhibited by administering an effective amount of one or more antibodies or molecules of the invention include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).

In specific embodiments, the present invention encompasses methods and compositions (e.g., antagonistic anti-B Lymphocyte Stimulator antibodies) for preventing, treating or ameliorating diseases or disorders associated with hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some immunodeficiency syndromes). In other specific embodiments, the present invention encompasses methods and compositions (e.g., agonistic anti-B Lymphocyte Stimulator antibodies) for preventing, treating or ameliorating diseases or disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency syndrome).

Autoimmune disorders, diseases, or conditions that may be detected, diagnosed, prognosed, or monitored using the antibodies of the invention include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjögren's syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulomatous, degenerative, and atrophic disorders).

Immunodeficiencies that may be detected, diagnosed, prognosed, or monitored using the antibodies of the invention include, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency.

DEFINITIONS

The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. Examples of molecules which are described by the term “antibody” in this application include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab′ fragments, F(ab′)₂, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain. The term “single chain Fv” or “scFv” as used herein refers to a polypeptide comprising a VL domain of antibody linked to a VH domain of an antibody. Antibodies that immunospecifically bind to B Lymphocyte Stimulator may have cross-reactivity with other antigens. Preferably, antibodies that immunospecifically bind to B Lymphocyte Stimulator do not cross-react with other antigens. Antibodies that immunospecifically bind to B Lymphocyte Stimulator can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass of immunoglobulin molecule.

Antibodies of the invention may also include multimeric forms of antibodies. For example, antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab′)₂ fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers withon an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities. Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509-7514, which is hereby incorporated by reference in its entirety. Antibody homodimers can be converted to Fab′2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby incorporated by reference in its entirety.

Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the J chain polypeptide. Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules. (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94, both of which are hereby incorporated by reference in their entireties.) ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971 which is hereby incorporated by reference in its entirety. Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

Unless otherwise defined in the specification, specific binding or immunospecifc binding by an anti-B Lymphocyte Stimulator antibody means that the anti-B Lymphocyte Stimulator antibody binds B Lymphocyte Stimulator but does not significantly bind to (i.e., cross react with) proteins other than B Lymphocyte Stimulator, such as other proteins in the same family of proteins, e.g., other TNF family ligands). An antibody that binds B Lymphocyte Stimulator protein and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the B Lymphocyte Stimulator-specific antibody of the invention preferentially binds B Lymphocyte Stimulator compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment. It is well known that the portion of a protein bound by an antibody is known as the epitope. An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein). Given that B Lymphocyte Stimulator-specific antibodies bind to epitopes of B Lymphocyte Stimulator, an antibody that specifically binds B Lymphocyte Stimulator may or may not bind fragments of B Lymphocyte Stimulator and/or variants of B Lymphocyte Stimulator (e.g., proteins that are at least 90% identical to B Lymphocyte Stimulator) depending on the presence or absence of the epitope bound by a given B Lymphocyte Stimulator-specific antibody in the B Lymphocyte Stimulator fragment or variant. Likewise, B Lymphocyte Stimulator-specific antibodies of the invention may bind species orthologues of B Lymphocyte Stimulator (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologue. Additionally, B Lymphocyte Stimulator-specific antibodies of the invention may bind modified forms of B Lymphocyte Stimulator, for example, B Lymphocyte Stimulator fusion proteins. In such a case when antibodies of the invention bind B Lymphocyte Stimulator fusion proteins, the antibody must make binding contact with the B Lymphocyte Stimulator moiety of the fusion protein in order for the binding to be specific. Antibodies that specifically bind to B Lymphocyte Stimulator can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

Furthermore, in the present application certain antibodies may be specific for either the membrane bound form of B Lymphocyte Stimulator, or the soluble form of B Lymphocyte Stimulator (i.e., 134-285 of SEQ ID NO:2, preferably trimers of proteins consisting of amino acids 134-285 of SEQ ID NO:2), or both. Antibodies of the present invention are defined as able to bind the membrane bound and/or soluble forms of B Lymphocyte Stimulator according to the assays described in Examples 1 through 19.

Preferably, an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of those referred to in Table 1, or a fragment or variant thereof.

An antibody of the invention “which binds the soluble form of B Lymphocyte Stimulator” is one which binds the 152 amino acid soluble form of the B Lymphocyte Stimulator protein (amino acids 134-285 of SEQ ID NO:3228). In specific embodiments of the invention, an antibody of the invention “which binds the soluble form of B Lymphocyte Stimulator” does not also bind the membrane-bound or membrane-associated form of B Lymphocyte Stimulator. Assays which measure binding to the soluble form of B Lymphocyte Stimulator include, but are not limited to, receptor binding inhibition assay or capture of soluble B Lymphocyte Stimulator from solution as described in Examples 8 and 9.

An antibody of the invention “which binds the membrane-bound form of B Lymphocyte Stimulator” is one which binds the membrane-associated (uncleaved) B Lymphocyte Stimulator protein. In specific embodiments of the invention, an antibody of the invention “which binds the membrane-bound form of B Lymphocyte Stimulator” does not also bind the soluble form of B Lymphocyte Stimulator. Binding to HIS-tagged B Lymphocyte Stimulator (as described herein) in an ELISA is an indicator that an antibody binds the membrane-bound form of B Lymphocyte Stimulator, but should not be relied upon as proof of specificity for the membrane-bound form of B Lymphocyte Stimulator. Assays that may be relied upon as proof of an antibody's specificity for membrane-bound B Lymphocyte Stimulator, include, but are not limited to, binding to plasma membranes expressing B Lymphocyte Stimulator as described in Example 2. An antibody of the invention “which binds the both the soluble form and the membrane-bound form of B Lymphocyte Stimulator” is one which binds both the membrane-bound form and the soluble form of B Lymphocyte Stimulator.

The term “variant” as used herein refers to a polypeptide that possesses a similar or identical function as a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof, but does not necessarily comprise a similar or identical amino acid sequence of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof, or possess a similar or identical structure of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof. A variant having a similar amino acid refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those referred to in Table 1) described herein; (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding a B Lymphocyte Stimulator polypeptide (e.g., SEQ ID NO:3228), a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those referred to in Table 1), described herein, of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, identical to the nucleotide sequence encoding a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those referred to in Table 1), described herein. A polypeptide with similar structure to a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quarternary structure of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody, or antibody fragment thereof, described herein. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.

To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions×100%). In one embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). The BLASTn and BLASTx programs of Altschul, et al. J. Mol. Biol. 215:403-410 (1990) have incorporated such an alogrithm. BLAST nucleotide searches can be performed with the BLASTn program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3389-3402 (1997). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. (See http://www.ncbi.nlm.nih.gov.)

Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an alogrithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti Comput. Appl. Biosci., 10:3-5 (1994); and FASTA described in Pearson and Lipman Proc. Natl. Acad. Sci. 85:2444-8 (1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

The term “derivative” as used herein, refers to a variant polypeptide of the invention that comprises, or alternatively consists of, an amino acid sequence of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term “derivative” as used herein also refers to a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an antibody that immunospecifically binds to B Lymphocyte Stimulator which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator antibody, may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator antibody, may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator antibody, may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator antibody, described herein.

The term “epitopes” as used herein refers to portions of B Lymphocyte Stimulator having antigenic or immunogenic activity in an animal, preferably a mammal. An epitope having immunogenic activity is a portion of B Lymphocyte Stimulator that elicits an antibody response in an animal. An eptiope having antigenic activity is a portion of B Lymphocyte Stimulator to which an antibody immunospecifically binds as determined by any method known in the art, for example, by the immunoassays described herein. Antigenic epitopes need not necessarily be immunogenic.

The term “fragment” as used herein refers to a polypeptide comprising an amino acid sequence of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues, of the amino acid sequence of B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator antibody (including molecules such as scFv's, that comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically binds to B Lymphocyte Stimulator.

The term “fusion protein” as used herein refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of an anti-B Lymphocyte Stimulator antibody of the invention and an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain).

The term “host cell” as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

By “isolated antibody” is intended an antibody removed from its native environment. Thus, an antibody produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1. ELISA results for three scFvs, I006E07, I008D05 and I016F04, that immunospecifically bind to U937 membranes, but not to bind to or cross-react with TNF-alpha or BSA.

FIG. 2. The results for three scFvs, I016H07, I001C09 and I018D07, in a receptor inhibition assay.

FIG. 3. ELISA results for two scFvs (I022D01 and I031F02) demonstrating their ability to bind to human B Lymphocyte Stimulator and to cross-react with mouse B Lymphocyte Stimulator, but not to bind to or cross-react with other antigens of the TNF ligand family.

FIG. 4. ELISA results for three scFvs (I031F09, I050A12, and I051C04) binding to U937 plasma membranes when either B Lymphocyte Stimulator or TNF-alpha is used as a competitor.

FIG. 5. Kinetic analysis of scFv antibody I003C02. A dilution series of I003C02 from 3 nM to 825 nM is shown. Association and dissociation curves were generated using a BIAcore 2000 and BIAevaluation 3.0 software.

FIG. 6. Typical titration curves for two scFv antibodies (I007F11 and I050A07) are shown in FIG. 6. Unlabelled B Lymphocyte Stimulator competed for binding to its receptor with an IC₅₀ value of 0.8 nM. The IC₅₀ values for I007F11 and I050A07 are 7.9 nM and 17.1 nM, respectively. The assay was performed in triplicate and standard error bars are shown.

FIG. 7. ELISA results for three scFvs clones (I074B12, I075F12 and I075A02) that immunospecifically bind to immobilized B Lymphocyte Stimulator, but not to U937 plasma membranes, TNF-alpha or BSA. As a control, a phage antibody that recognizes TNFα, is also shown in FIG. 7.

FIG. 8. The results for two scFvs (I025B09 and I026C04) in a receptor inhibition assay.

FIG. 9. ELISA results for two scFvs clones (I067F05 and I078D02) demonstrating their ability to bind to immobilized human B Lymphocyte Stimulator and to cross-react with immobilized mouse B Lymphocyte Stimulator, but not to bind to or cross-react with other antigens of the TNF ligand family.

As a control, a phage antibody that recognizes TNFα, is also shown in FIG. 7.

FIG. 10. Kinetic analysis of scFV antibody I002A01. A dilution series of I002A01 from 3 nM to 1650 nM is shown. Association and dissociation curves were generated using a BIAcore 2000 and BIAevaluation 3.0 software.

FIG. 11. Typical titration curves for two scFvs, I0068C06 and I074B12, are shown in FIG. 11. Unlabelled B Lymphocyte Stimulator competed for binding to its receptor with an inhibitory constant 50 (IC₅₀) value of 0.66 nM. The IC₅₀ values for I0068C06 and I074B12 are 61 nM and 13 nM, respectively. The assay was performed in triplicate and standard error bars are shown.

FIG. 12. ELISA results for three clones (I079C01, I081C10 and I082A02) demonstrating their ability to bind histidine-tagged B Lymphocyte Stimulator, U937 plasma membranes, but not to bind immobilized biotinylated B Lymphocyte Stimulator.

FIG. 13. ELISA results for three scFvs (I079B04, I079F08, and I080B01) binding to U937 plasma membranes when either histidine-tagged B Lymphocyte Stimulator or biotinylated B Lymphocyte Stimulator is used as a competitor.

FIG. 14. An example of the dissociation section of a typical sensorgram for 8 scFvs is shown in FIG. 14. An anti-TNFα antibody that does not recognize B Lymphocyte Stimulator was included as a control. Of the 8 scFvs exemplified, I079F06 was identified for further study due to the relatively high numbers of RU's bound to the surface.

FIG. 15. A typical example of the binding curves generated for the scFv antibody I082C03 is shown in FIG. 15. The off-rate for this clone was calculated as 2×10⁻³ s⁻¹. The affinity of I082C03 was calculated as 20 nM, assuming 100% activity of the scFv.

FIG. 16. ELISA results for three scFvs (I079B04, I079F08, and I080B01) binding to P388 plasma membranes when either histidine-tagged B Lymphocyte Stimulator or biotinylated B Lymphocyte Stimulator is used as a competitor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator or a fragment or variant of B Lymphocyte Stimulator. In particular, the invention provides antibodies such as, for example, single chain Fvs (scFvs) having an amino acid sequence of any one of SEQ ID NOS:1-2128, as referred to in Table 1. In particular, the present invention encompasses antibodies that immunospecifically bind to a polypeptide, a polypeptide fragment or variant, or an epitope of human B Lymphocyte Stimulator (SEQ ID NOS:3228 and/or 3229) or B Lymphocyte Stimulator expressed on human monocytes; murine B Lymphocyte Stimulator (SEQ ID NOS:3230 and/or 3231) or B Lymphocyte Stimulator expressed on murine monocytes; rat B Lymphocyte Stimulator (either the soluble forms as given in SEQ ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey B Lymphocyte Stimulator (e.g., the monkey B Lymphocyte Stimulator polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey B Lymphocyte Stimulator, or B Lymphocyte Stimulator expressed on monkey monocytes) (as determined by immunoassays known in the art for assaying specific antibody-antigen binding).

The polypeptide sequence shown in SEQ ID NO:3228 was obtained by sequencing and translating the cDNA of the HNEDU15 clone which was deposited on Oct. 22, 1996 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, and assigned ATCC™ Accession No. 97768. The deposited clone is contained in the pBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.). The ATCC™ deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

The polypeptide sequence shown in SEQ ID NO:3229 was obtained by sequencing and translating the cDNA of the HDPMC52 clone, which was deposited on Dec. 10, 1998 at the American Type Culture Collection, and assigned ATCC™ Accession No. 203518. The deposited clone is contained in the pBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.). The ATCC™ deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

The B Lymphocyte Stimulator polypeptides bound by the antibodies of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to antibodies that bind monomers and multimers of the B Lymphocyte Stimulator polypeptides of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them. In specific embodiments, the antibodies of the invention bind B Lymphocyte Stimulator monomers, dimers, trimers or tetramers. In additional embodiments, the antibodies of the invention bind at least dimers, at least trimers, or at least tetramers of B Lymphocyte Stimulator.

Multimeric B Lymphocyte Stimulator bound by the antibodies of the invention may be homomers or heteromers. A B Lymphocyte Stimulator homomer, refers to a multimer containing only B Lymphocyte Stimulator polypeptides (including B Lymphocyte Stimulator fragments, variants, and fusion proteins, as described herein). These homomers may contain B Lymphocyte Stimulator polypeptides having identical or different amino acid sequences. In specific embodiments, the antibodies of the invention bind a B Lymphocyte Stimulator homodimer (e.g., containing two B Lymphocyte Stimulator polypeptides having identical or different amino acid sequences) or a B Lymphocyte Stimulator homotrimer (e.g., containing three B Lymphocyte Stimulator polypeptides having identical or different amino acid sequences). In a preferred embodiment, the antibodies of the invention bind homotrimers of B Lymphocyte Stimulator. In additional embodiments, the antibodies of the invention bind a homomeric B Lymphocyte Stimulator multimer which is at least a homodimer, at least a homotrimer, or at least a homotetramer.

Heteromeric B Lymphocyte Stimulator refers to a multimer containing heterologous polypeptides (i.e., polypeptides of a different protein) in addition to the B Lymphocyte Stimulator polypeptides of the invention. In a specific embodiment, the antibodies of the invention bind a B Lymphocyte Stimulator heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the antibodies of the invention bind a heteromeric B Lymphocyte Stimulator multimer which is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer. In highly preferred embodiments, the antibodies of the invention bind a heterotrimer comprising both B Lymphocyte Stimulator polypeptides and APRIL polypeptides (SEQ ID NO:3239; GenBank Accession No. AF046888; PCT International Publication Number WO97/33902; J. Exp. Med. 188(6):1185-1190) or fragments or variants thereof. In other highly preferred embodiments, the antibodies of the invention bind a heterotrimer comprising one B Lymphocyte Stimulator polypeptide (including fragments or variants) and two APRIL polypeptides (including fragments or variants). In still other highly preferred embodiments, the antibodies of the invention bind a heterotrimer comprising two B Lymphocyte Stimulator polypeptides (including fragments or variants) and one APRIL polypeptide (including fragments or variants). In a further nonexclusive embodiment, the heteromers bound by the antibodies of the invention contain CD40 ligand polypeptide sequence(s), or biologically active fragment(s) or variant(s) thereof.

In particularly preferred embodiments, the antibodies of the invention bind homomeric, especially homotrimeric, B Lymphocyte Stimulator polypeptides, wherein the individual protein components of the multimers consist of the mature form of B Lymphocyte Stimulator (e.g., amino acids residues 134-285 of SEQ ID NO:3228, or amino acids residues 134-266 of SEQ ID NO:3229) or fragments or variants thereof. In other specific embodiments, antibodies of the invention bind heteromeric, especially heterotrimeric, B Lymphocyte Stimulator polypeptides such as a heterotrimer containing two B Lymphocyte Stimulator polypeptides and one APRIL polypeptide or a heterotrimer containing one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides, and wherein the individual protein components of the B Lymphocyte Stimulator heteromer consist of the mature extracellular soluble portion of either B Lymphocyte Stimulator (e.g., amino acids residues 134-285 of SEQ ID NO:3228, or amino acids residues 134-266 of SEQ ID NO:3229) or fragments or variants thereof, or the mature extracellular soluble portion APRIL (e.g., amino acid residues 105-250 of SEQ ID NO:3239) or fragments or variants thereof.

In specific embodiments, the antibodies of the invention bind conformational epitopes of a B Lymphocyte Stimulator monomeric protein. In specific embodiments, the antibodies of the invention bind conformational epitopes of a B Lymphocyte Stimulator multimeric, especially trimeric, protein. In other embodiments, antibodies of the invention bind conformational epitopes that arise from the juxtaposition of B Lymphocyte Stimulator with a heterologous polypeptide, such as might be present when B Lymphocyte Stimulator forms heterotrimers (e.g., with APRIL polypeptides (e.g., SEQ ID SEQ ID NO:3239)), or in fusion proteins between B Lymphocyte Stimulator and a heterologous polypeptide.

In a specific embodiment, antibodies of the invention that specifically bind heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide, comprise all or a portion of SEQ ID NOS: 1881 or 1884 (e.g., one or more CDR regions, a VH domain or a VL domain). In a specific embodiment, the heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide comprise two B Lymphocyte Stimulator polypeptides and one APRIL polypeptide. In a specific embodiment, the heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide comprise one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides.

In a specific embodiment, antibodies of the invention that specifically bind heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide, comprise all or a portion of any one of SEQ ID NOS: 3240-3247 (e.g., one or more CDR regions, a VH domain or a VL domain). The sequences of SEQ ID NOS: 3240-3247 are presented after Table 1 just prior to the claims. In a specific embodiment, the heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide comprise two B Lymphocyte Stimulator polypeptides and one APRIL polypeptide. In a specific embodiment, the heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide comprise one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides.

B Lymphocyte Stimulator multimers bound by the antibodies of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, B Lymphocyte Stimulator multimers, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, B Lymphocyte Stimulator heteromultimers, such as, for example, B Lymphocyte Stimulator heterotrimers or B Lymphocyte Stimulator heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, B Lymphocyte Stimulator multimers are formed by covalent associations with and/or between the B Lymphocyte Stimulator polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:3228 or SEQ ID NO:3229). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a B Lymphocyte Stimulator fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a B Lymphocyte Stimulator-Fc fusion protein. In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from CD40L, or a soluble fragment thereof. In another embodiment, two or B Lymphocyte Stimulator polypeptides are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple B Lymphocyte Stimulator polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.

In one embodiment, antibodies of the invention immunospecifically bind a B Lymphocyte Stimulator polypeptide having the amino acid sequence of SEQ ID NO:3228 or as encoded by the cDNA clone contained in ATCC™ No. 97768, or a polypeptide comprising a portion (i.e., a fragment) of the above polypeptides. In another embodiment, the invention provides an antibody that binds an isolated B Lymphocyte Stimulator polypeptide having the amino acid sequence of SEQ ID NO:3229 or the amino acid sequence encoded by the cDNA clone contained in ATCC™ No. 203518, or an antibody that binds polypeptide comprising a portion (i.e, fragment) of the above polypeptides.

Antibodies of the invention that bind B Lymphocyte Stimulator polypeptides may bind them in as isolated polypeptides, in their naturally occurring state and/or their native conformation. By “isolated polypeptide” is intended a polypeptide removed from its native environment. Thus, a polypeptide produced by and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an “isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host cell. Thus, antibodies of the present invention may bind recombinantly produced B Lymphocyte Stimulator polypeptides.

Antibodies of the present invention may also bind B Lymphocyte Stimulator expressed on the surface of a cell, wherein said B Lymphocyte Stimulator polypeptide is encoded by a polynucleotide encoding amino acids 1 to 285 of SEQ ID NO:2 operably associated with a regulatory sequence that controls expression of said polynucleotide. In certain embodiments, said B Lymphocyte Stimulator polypeptide expressed on the surface of a cell is a recombinant B Lymphocyte Stimulator polypeptide. In other embodiments, said B Lymphocyte Stimulator polypeptide expressed on the surface of the cell is a naturally occurring B Lymphocyte Stimulator polypeptide. As a non-limiting example, an antibody of the invention may bind a B Lymphocyte Stimulator expressed on the surface of the cell wherein Lys-132 and/or Arg-133 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, thereby preventing or diminishing release of the soluble form of B Lymphocyte Stimulator from cells expressing B Lymphocyte Stimulator.

Antibodies of the present invention may also bind B Lymphocyte Stimulator secreted by a cell, wherein said B Lymphocyte Stimulator polypeptide is encoded by a polynucleotide encoding amino acids 1 to 285 of SEQ ID NO:2 operably associated with a regulatory sequence that controls expression of said polynucleotide. In certain embodiments, said B Lymphocyte Stimulator polypeptide secreted by a cell is a recombinant B Lymphocyte Stimulator polypeptide. In other embodiments, said B Lymphocyte Stimulator polypeptide secreted by a cell is a naturally occurring B Lymphocyte Stimulator polypeptide.

Antibodies of the present invention immunospecifically bind to polypeptides comprising or alternatively, consisting of, the amino acid sequence of SEQ ID NO:3228, encoded by the cDNA contained in the plasmid having ATCC™ accession number 97768, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone. Antibodies of the present invention also bind to fragments of the amino acid sequence of SEQ ID NO:3228, encoded by the cDNA contained in the plasmid having ATCC™ accession number 97768, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone.

Additionally, antibodies of the present invention bind polypeptides comprising or alternatively, consisting of, the amino acid sequence of SEQ ID NO:3229, encoded by the cDNA contained in the plasmid having ATCC™ accession number 203518, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone. Antibodies of the present invention also bind to fragments of the amino acid sequence of SEQ ID NO:3229, encoded by the cDNA contained in the plasmid having ATCC™ accession number 203518, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone.

In addition, antibodies of the invention bind polypeptides or polypeptide fragments comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NOS: 3230 through 3237.

In specific embodiments, the antibodies of the present invention immunospecifically bind polypeptide fragments including polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:3228, encoded by the cDNA contained in the deposited clone, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone. Protein fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments that may be bound by the antibodies of the present invention, include, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250, and/or 251 to 285 of SEQ ID NO:3228. Moreover, polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length.

In specific embodiments, antibodies of the present invention bind polypeptide fragments comprising, or alternatively consisting of, amino acid residues: 1-46, 31-44, 47-72, 73-285, 73-83, 94-102, 148-152, 166-181, 185-209, 210-221, 226-237, 244-249, 253-265, and/or 277-285 of SEQ ID NO:3228. In a specific embodiment, antibodies of the invention bind an epitope comprising amino acids 165-171 of SEQ ID NO:3228. In another embodiment, the CDRs of antibodies of the invention make contacts with one or more amino acids in the sequence of amino acids 165-171 of SEQ ID NO:3228. In another embodiment, antibodies of the invention whose CDRs make contact with one or more amino acids in the sequence of amino acids 165-171 of SEQ ID NO:3228 disrupt B Lymphocyte Stimulator-B Lymphocyte Stimulator receptor interactions.

It will be recognized by one of ordinary skill in the art that mutations targeted to regions of a B Lymphocyte Stimulator polypeptide of SEQ ID NO:3228 which encompass the nineteen amino acid residue insertion which is not found in the B Lymphocyte Stimulator polypeptide sequence of SEQ ID NO:3229 (i.e., amino acid residues Val-142 through Lys-160 of the sequence of SEQ ID NO:3229) may affect the observed biological activities of the B Lymphocyte Stimulator polypeptide. More specifically, a partial, non-limiting and non-exclusive list of such residues of the B Lymphocyte Stimulator polypeptide sequence which may be targeted for mutation includes the following amino acid residues of the B Lymphocyte Stimulator polypeptide sequence as shown in SEQ ID NO:3228: V-142; T-143; Q-144; D-145; C-146; L-147; Q-148; L-149; I-150; A-151; D-152; S-153; E-154; T-155; P-156; T-157; I-158; Q-159; and K-160. Thus, in specific embodiments, antibodies of the present invention that bind B Lymphocyte Stimulator polypeptides which have one or more mutations in the region from V-142 through K-160 of SEQ ID NO:3228 are contemplated.

Polypeptide fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments that may be bound by antibodies of the present invention, include, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 15, 16-30, 31-46, 47-55, 56-72, 73-104, 105-163, 163-188, 186-210 and 210-284 of the amino acid sequence disclosed in SEQ ID NO:3228. Additional representative examples of polypeptide fragments that may be bound by antibodies of the present invention, include, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 143, 1-150, 47-143, 47-150, 73-143, 73-150, 100-150, 140-145, 142-148, 140-150, 140-200, 140-225, and 140-266 of the amino acid sequence disclosed in SEQ ID NO:3229. Moreover, polypeptide fragments that may be bound by antibodies of the present invention, can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length. In this context, “about” means the particularly recited ranges and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid residues at either or both the amino- and carboxy-termini.

Additional preferred embodiments encompass antibodies that bind polypeptide fragments comprising, or alternatively consisting of, the predicted intracellular domain of B Lymphocyte Stimulator (e.g., amino acid residues 1-46 of SEQ ID NO:3228), the predicted transmembrane domain of B Lymphocyte Stimulator (e.g., amino acid residues 47-72 of SEQ ID NO:3228), the predicted extracellular domain of B Lymphocyte Stimulator (e.g., amino acid residues 73-285 of SEQ ID NO:3228), the mature soluble extracellular domain of B Lymphocyte Stimulator (e.g., amino acids residues 134-285 of SEQ ID NO:3228), the predicted TNF conserved domain of B Lymphocyte Stimulator (e.g., amino acids 191 to 284 of SEQ ID NO:3228), and a polypeptide comprising, or alternatively, consisting of the predicted intracellular domain fused to the predicted extracellular domain of B Lymphocyte Stimulator (amino acid residues 1-46 fused to amino acid residues 73-285 of SEQ ID NO:3228).

Further additional preferred embodiments encompass polypeptide fragments comprising, or alternatively consisting of, the predicted intracellular domain of B Lymphocyte Stimulator (amino acid residues 1-46 of SEQ ID NO:3229), the predicted transmembrane domain of B Lymphocyte Stimulator (amino acid residues 47-72 of SEQ ID NO:3229), the predicted extracellular domain of B Lymphocyte Stimulator (amino acid residues 73-266 of SEQ ID NO:3229), the predicted TNF conserved domain of B Lymphocyte Stimulator (amino acids 172 to 265 of SEQ ID NO:3229), and a polypeptide comprising, or alternatively, consisting of the predicted intracellular domain fused to the predicted extracellular domain of B Lymphocyte Stimulator (amino acid residues 1-46 fused to amino acid residues 73-266 of SEQ ID NO:3229).

Certain additional embodiments of the invention encompass antibodies that bind polypeptide fragments comprising, or alternatively consisting of, the predicted beta-pleated sheet regions of the B Lymphocyte Stimulator polypeptides of SEQ ID NO:3228 and SEQ ID NO:3229. These polypeptide fragments comprising the beta-pleated sheets of B Lymphocyte Stimulator comprise, or alternatively consist of, amino acid residues Gln-144 to Ala-151, Phe-172 to Lys-173, Ala-177 to Glu-179, Asn-183 to Ile-185, Gly-191 to Lys-204, His-210 to Val-219, Leu-226 to Pro-237, Asn-242 to Ala-251, Gly-256 to Ile-263 and/or Val-276 to Leu-284 of SEQ ID NO:3228. In another, nonexclusive embodiment, these polypeptide fragments comprising the beta-pleated sheets of B Lymphocyte Stimulator comprise, or alternatively consist of, amino acid residues Phe-153 to Lys-154, Ala-158 to Glu-160, Asn-164 to Ile-166, Gly-172 to Lys-185, His-191 to Val-200, Leu-207 to Pro-218, Asn-223 to Ala-232, Gly-237 to Ile-244 and/or Val-257 to Leu-265 of SEQ ID NO:3229.

A partial, non-limiting, and exemplary list of polypeptides that may be bound by the antibodies of the invention includes polypeptides that comprise, or alternatively consist of, combinations of amino acid sequences of the invention includes, for example, [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Val-199 to Ala-248] fused to [Gly-249 to Leu-285] of SEQ ID NO:3228; or [Met-1 to Lys-113] fused to [Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Val-199 to Ala-248] fused to [Gly-249 to Leu-285] of SEQ ID NO:3228; or [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Gly-249 to Leu-285] of SEQ ID NO:3228. Other combinations of amino acids sequences that may be bound by the antibodies of the invention may include the polypeptide fragments in an order other than that recited above (e.g., [Leu-114 to Thr-141] fused to [Val-199 to Ala-248] fused to [Gly-249 to Leu-285] fused to [Val-142 to Lys-160] of (SEQ ID NO:3228). Other combinations of amino acids sequences that may be bound by the antibodies of the invention may also include heterologous polypeptide fragments as described herein and/or other polypeptides or polypeptide fragments of the present invention (e.g., [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Gly-249 to Leu-285] of SEQ ID NO:3228 fused to a FLAG tag; or [Met-1 to Lys-113] of SEQ ID NO:3228 fused to [Leu-114 to Thr-141] of SEQ ID NO:3228 fused to [Glu-135 to Asn-165] of SEQ ID NO:39 fused to [Val-142 to Lys-160] of SEQ ID NO:3228 fused to [Gly-161 to Gln-198] of SEQ ID NO:3228 fused to [Val-199 to Ala-248] of SEQ ID NO:3228 fused to [Gly-249 to Leu-285] of SEQ ID NO:3228).

A partial, non-limiting, and exemplary list of polypeptides that may be bound by the antibodies of the invention includes polypeptides that comprise, or alternatively consist of, combinations of amino acid sequences includes, for example, [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-266] of SEQ ID NO:3229; [Met-1 to Lys-113] fused to [Gly-142 to Gln-179] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-266] of SEQ ID NO:3229; or [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to [Gly-230 to Leu-266] of SEQ ID NO:3229. Other of amino acids sequences that may be bound by the antibodies of the invention combinations may include the polypeptide fragments in an order other than that recited above (e.g., [Leu-114 to Thr-141] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-266] fused to [Gly-142 to Gln-179] of SEQ ID NO:3229). Other combinations of amino acid sequences that may be bound by the antibodies of the invention may also include heterologous polypeptide fragments as described herein and/or other polypeptides or polypeptide fragments of the present invention (e.g., [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to [Gly-230 to Leu-266] of SEQ ID NO:3229 fused to a FLAG tag (SEQ ID NO:3238) or, [Met-1 to Lys-113] of SEQ ID NO:3229 fused to [Leu-114 to Thr-141] of SEQ ID NO:3229 fused to [Glu-135 to Asn-165] of SEQ ID NO:39 fused to [Gly-142 to Gln-179] of SEQ ID NO:3229 fused to [Val-180 to Ala-229] of SEQ ID NO:3229 fused to [Gly-230 to Leu-266] of SEQ ID NO:3229.

Additional embodiments of the invention encompass antibodies that bind B Lymphocyte Stimulator polypeptide fragments comprising, or alternatively consisting of, functional regions of polypeptides of the invention, such as the Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Emini surface-forming regions and Jameson-Wolf regions of high antigenic index set out in Tables 9 and 10 and as described herein. In a preferred embodiment, the polypeptide fragments bound by the antibodies of the invention are antigenic (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) of a complete (i.e., full-length) B Lymphocyte Stimulator polypeptide (e.g., SEQ ID NOS:3228 and 3229).

The data representing the structural or functional attributes of the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3228 (Table 9) or the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3229 (Table 10), as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. Column I represents the results of a Garnier-Robson analysis of alpha helical regions; Column II represents the results of a Chou-Fasman analysis of alpha helical regions; Column III represents the results of a Garnier Robson analysis of beta sheet regions; Column IV represents the results of a Chou-Fasman analysis of beta sheet regions; Column V represents the results of a Garnier Robson analysis of turn regions; Column VI represents the results of a Chou-Fasman analysis of turn regions; Column VII represents the results of a Garnier Robson analysis of coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot; Column IX represents a Hopp-Woods hydrophobicity plot; Column X represents the results of an Eisenberg analysis of alpha amphipathic regions; Column XI represents the results of an Eisenberg analysis of beta amphipathic regions; Column XII represents the results of a Karplus-Schultz analysis of flexible regions; Column XIII represents the Jameson-Wolf antigenic index score; and Column XIV represents the Emini surface probability plot.

In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Tables 9 and 10 can be used to determine regions of the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3228 (Table 9) or the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3229 (Table 10) which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

The above-mentioned preferred regions set out in Tables 9 and 10 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID NO:2. As set out in Tables 9 and 10, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Preferably, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides or B Lymphocyte Stimulator polypeptide fragments and variants comprising regions of B Lymphocyte Stimulator that combine several structural features, such as several (e.g., 1, 2, 3, or 4) of the same or different region features set out above and in Tables 9 and 10.

TABLE 9 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A . . . . . . 0.73 −0.71 . . . 0.95 1.39 Asp 2 A . . . . T . 1.12 −0.66 * . . 1.15 1.56 Asp 3 A . . . . T . 1.62 −1.09 * . . 1.15 2.12 Ser 4 A . . . . T . 2.01 −1.51 . . . 1.15 4.19 Thr 5 A . . . . T . 2.40 −2.13 . . F 1.30 4.35 Glu 6 A A . . . . . 2.70 −1.73 * * F 0.90 4.51 Arg 7 A A . . . . . 2.81 −1.34 * * F 0.90 4.51 Glu 8 A A . . . . . 2.00 −1.73 * * F 0.90 6.12 Gln 9 A A . . . . . 1.99 −1.53 * * F 0.90 2.91 Ser 10 A . . B . . . 2.00 −1.04 * * F 0.90 2.15 Arg 11 A . . B . . . 1.33 −0.66 * * F 0.90 1.66 Leu 12 A . . B . . . 0.41 −0.09 * * F 0.45 0.51 Thr 13 A . . B . . . 0.46 0.20 * * F −0.15 0.32 Ser 14 A A . . . . . 0.50 −0.19 * * . 0.30 0.32 Cys 15 A A . . . . . 0.91 −0.19 * * . 0.30 0.78 Leu 16 A A . . . . . 0.80 −0.87 * * F 0.90 1.06 Lys 17 A A . . . . . 1.61 −1.36 . * F 0.90 1.37 Lys 18 A A . . . . . 1.32 −1.74 . * F 0.90 4.44 Arg 19 A A . . . . . 1.67 −1.70 . * F 0.90 5.33 Glu 20 A A . . . . . 1.52 −2.39 . * F 0.90 5.33 Glu 21 A A . . . . . 2.38 −1.70 . * F 0.90 2.20 Met 22 A A . . . . . 2.33 −1.70 . * F 0.90 2.24 Lys 23 A A . . . . . 1.62 −1.70 * * F 0.90 2.24 Leu 24 A A . . . . . 0.66 −1.13 * * F 0.75 0.69 Lys 25 A A . . . . . 0.36 −0.49 . * F 0.45 0.52 Glu 26 A A . B . . . −0.53 −0.71 * * . 0.60 0.35 Cys 27 A A . B . . . −0.74 −0.03 * * . 0.30 0.30 Val 28 A A . B . . . −1.00 −0.03 * * . 0.30 0.12 Ser 29 A A . B . . . −0.08 0.40 * * . −0.30 0.11 Ile 30 A . . B . . . −0.08 0.40 * * . −0.30 0.40 Leu 31 A . . B . . . −0.08 −0.17 * . . 0.45 1.08 Pro 32 . . . B . . C 0.29 −0.81 * . F 1.10 1.39 Arg 33 . . . . T . . 0.93 −0.81 . * F 1.50 2.66 Lys 34 . . . . T . . 0.93 −1.07 . . F 1.84 4.98 Glu 35 . . . . . . C 0.97 −1.37 * * F 1.98 4.32 Ser 36 . . . . . T C 1.89 −1.16 * * F 2.52 1.64 Pro 37 . . . . . T C 1.80 −1.16 * * F 2.86 1.60 Ser 38 . . . . T T . 1.39 −0.77 * . F 3.40 1.24 Val 39 A . . . . T . 1.39 −0.39 . * F 2.36 1.24 Arg 40 A . . . . . . 1.39 −0.77 * * F 2.46 1.60 Ser 41 A . . . . . . 1.34 −1.20 * * F 2.46 2.00 Ser 42 . . . . T T . 1.60 −1.16 . * F 3.06 2.67 Lys 43 . . . . T T . 1.09 −1.80 . * F 3.06 2.72 Asp 44 . . . . T T . 1.13 −1.11 * * F 3.40 1.67 Gly 45 A . . . . T . 0.43 −0.81 * * F 2.66 1.03 Lys 46 A A . . . . . 0.14 −0.70 . . F 1.77 0.52 Leu 47 A A . . . . . 0.13 −0.20 * . . 0.98 0.31 Leu 48 A A . . . . . −0.72 0.29 * . . 0.04 0.46 Ala 49 A A . . . . . −1.53 0.54 . * . −0.60 0.19 Ala 50 A A . . . . . −2.00 1.23 . . . −0.60 0.19 Thr 51 A A . . . . . −2.63 1.23 . . . −0.60 0.19 Leu 52 A A . . . . . −2.63 1.04 . . . −0.60 0.19 Leu 53 A A . . . . . −2.63 1.23 . . . −0.60 0.15 Leu 54 A A . . . . . −2.34 1.41 . . . −0.60 0.09 Ala 55 A A . . . . . −2.42 1.31 . . . −0.60 0.14 Leu 56 A A . . . . . −2.78 1.20 . . . −0.60 0.09 Leu 57 A . . . . T . −2.78 1.09 . . . −0.20 0.06 Ser 58 A . . . . T . −2.28 1.09 . . . −0.20 0.05 Cys 59 A . . . . T . −2.32 1.07 . . . −0.20 0.09 Cys 60 A . . . . T . −2.59 1.03 . . . −0.20 0.08 Leu 61 . . B B . . . −2.08 0.99 . . . −0.60 0.04 Thr 62 . . B B . . . −1.97 0.99 . . . −0.60 0.11 Val 63 . . B B . . . −1.91 1.20 . . . −0.60 0.17 Val 64 . . B B . . . −1.24 1.39 . . . −0.60 0.33 Ser 65 . . B B . . . −1.43 1.10 . . . −0.60 0.40 Phe 66 A . . B . . . −1.21 1.26 . . . −0.60 0.40 Tyr 67 A . . B . . . −1.49 1.11 . . . −0.60 0.54 Gln 68 A . . B . . . −1.44 0.97 . . . −0.60 0.41 Val 69 A . . B . . . −0.59 1.27 . . . −0.60 0.39 Ala 70 A . . B . . . −0.63 0.89 . . . −0.60 0.43 Ala 71 A . . B . . . 0.07 0.56 . * . −0.60 0.25 Leu 72 A . . . . T . −0.50 0.16 . * . 0.10 0.55 Gln 73 A . . . . T . −1.09 0.20 . . F 0.25 0.45 Gly 74 A . . . . T . −0.53 0.20 . . F 0.25 0.45 Asp 75 A . . . . T . −0.76 0.09 . * F 0.25 0.73 Leu 76 A A . . . . . −0.06 0.09 . * F −0.15 0.35 Ala 77 A A . . . . . 0.17 −0.31 . * . 0.30 0.69 Ser 78 A A . . . . . 0.17 −0.24 . * . 0.30 0.42 Leu 79 A A . . . . . −0.30 −0.24 . * . 0.30 0.88 Arg 80 A A . . . . . −0.30 −0.24 . * . 0.30 0.72 Ala 81 A A . . . . . 0.17 −0.34 . * . 0.30 0.93 Glu 82 A A . . . . . 0.72 −0.30 . * . 0.45 1.11 Leu 83 A A . . . . . 0.99 −0.49 . * . 0.30 0.77 Gln 84 A A . . . . . 1.21 0.01 . * . −0.15 1.04 Gly 85 A A . . . . . 1.10 0.01 * * . −0.30 0.61 His 86 A A . . . . . 1.73 0.01 * * . −0.15 1.27 His 87 A A . . . . . 0.92 −0.67 . * . 0.75 1.47 Ala 88 A A . . . . . 1.52 −0.39 . * . 0.45 1.22 Glu 89 A A . . . . . 0.93 −0.39 . . . 0.45 1.39 Lys 90 A A . . . . . 0.93 −0.39 * . F 0.60 1.03 Leu 91 A . . . . T . 0.38 −0.46 * . . 0.85 1.01 Pro 92 A . . . . T . 0.07 −0.46 . . . 0.70 0.59 Ala 93 A . . . . T . 0.07 −0.03 . . . 0.70 0.29 Gly 94 A . . . . T . −0.14 0.47 . . . −0.20 0.36 Ala 95 A . . . . . . −0.14 0.21 . * . −0.10 0.36 Gly 96 A . . . . . . 0.08 −0.21 . . F 0.65 0.71 Ala 97 A . . . . . . −0.06 −0.21 . . F 0.65 0.72 Pro 98 A . . . . . . −0.28 −0.21 . * F 0.65 0.71 Lys 99 A A . . . . . 0.07 −0.03 . . F 0.45 0.59 Ala 100 A A . . . . . 0.66 −0.46 . . F 0.60 1.01 Gly 101 A A . . . . . 0.41 −0.96 . . F 0.90 1.13 Leu 102 A A . . . . . 0.79 −0.89 . . F 0.75 0.57 Glu 103 A A . . . . . 0.41 −0.46 * . F 0.45 0.88 Glu 104 A A . . . . . −0.49 −0.46 * . F 0.45 0.89 Ala 105 A A . . . . . −0.21 −0.24 . . . 0.30 0.81 Pro 106 A A . . . . . −0.46 −0.44 . . . 0.30 0.67 Ala 107 A A . . . . . 0.01 0.06 . . . −0.30 0.39 Val 108 A A . . . . . −0.80 0.49 . * . −0.60 0.38 Thr 109 A A . . . . . −0.76 0.67 . * . −0.60 0.20 Ala 110 A A . . . . . −1.06 0.24 * * . −0.30 0.40 Gly 111 A A . . . . . −1.54 0.43 * * . −0.60 0.38 Leu 112 A A . . . . . −0.96 0.57 * * . −0.60 0.23 Lys 113 . A B . . . . −0.31 0.09 * * . −0.30 0.39 Ile 114 . A B . . . . −0.21 0.01 * . . −0.30 0.61 Phe 115 . A B . . . . −0.21 0.01 * . . 0.15 1.15 Glu 116 . A . . . . C −0.08 −0.17 * . F 1.25 0.58 Pro 117 . A . . . . C 0.39 0.26 * * F 1.10 1.28 Pro 118 . . . . . . C 0.34 −0.00 . . F 2.20 1.47 Ala 119 . . . . . T C 0.89 −0.79 . * F 3.00 1.47 Pro 120 . . . . . T C 1.59 −0.36 . * F 2.25 0.94 Gly 121 . . . . T T . 1.29 −0.39 . * F 2.15 0.98 Glu 122 . . . . T T . 1.20 −0.43 . . F 2.00 1.30 Gly 123 . . . . . . C 1.41 −0.54 . . F 1.60 1.12 Asn 124 . . . . . T C 2.00 −0.57 . . F 1.50 1.97 Ser 125 . . . . . T C 1.91 −0.60 . * F 1.50 1.82 Ser 126 . . . . . T C 2.37 −0.21 . * F 1.54 2.47 Gln 127 . . . . . T C 2.37 −0.64 . * F 2.18 3.01 Asn 128 . . . . . . C 2.76 −0.64 . . F 2.32 3.61 Ser 129 . . . . . T C 2.87 −1.03 . . F 2.86 5.39 Arg 130 . . . . T T . 2.58 −1.41 * . F 3.40 6.09 Asn 131 . . . . T T . 2.02 −1.31 * . F 3.06 3.83 Lys 132 . . . . T T . 2.02 −1.07 * . F 2.72 2.12 Arg 133 . . . . T . . 1.68 −1.06 * . F 2.18 1.88 Ala 134 . . . . . . C 1.77 −0.63 * . F 1.64 1.15 Val 135 . . . . . . C 1.66 −0.60 * . F 1.49 0.89 Gln 136 . . . . . . C 1.66 −0.60 * . F 1.83 0.79 Gly 137 . . . . . T C 1.30 −0.60 * . F 2.52 1.35 Pro 138 . . . . . T C 0.33 −0.61 * . F 2.86 2.63 Glu 139 . . . . T T . 0.61 −0.61 * . F 3.40 1.13 Glu 140 A . . . . T . 1.47 −0.53 * . F 2.66 1.64 Thr 141 A . . . . . . 1.47 −0.56 . . F 2.12 1.84 Val 142 A . . . . . . 1.14 −0.99 . . F 1.78 1.77 Thr 143 A . . . . T . 0.54 −0.41 . . F 1.19 0.55 Gln 144 A . . . . T . 0.54 0.27 * . F 0.25 0.31 Asp 145 A . . . . T . −0.27 0.19 * . F 0.25 0.73 Cys 146 A . . . . T . −0.84 0.23 * . . 0.10 0.42 Leu 147 A A . . . . . −0.58 0.43 * . . −0.60 0.17 Gln 148 A A . . . . . −0.27 0.53 * . . −0.60 0.10 Leu 149 A A . . . . . −0.57 0.53 * * . −0.30 0.32 Ile 150 A A . . . . . −0.57 0.34 * . . 0.30 0.52 Ala 151 . A . . . . C −0.21 −0.34 . * . 1.40 0.52 Asp 152 . . . . T T . 0.39 −0.26 . * F 2.45 0.91 Ser 153 . . . . . T C 0.08 −0.51 . . F 3.00 2.00 Glu 154 . . . . . T C −0.00 −0.71 . . F 2.70 2.86 Thr 155 . . . . . T C 0.89 −0.53 * . F 2.40 1.20 Pro 156 . . . B . . C 1.52 −0.13 * . F 1.56 1.55 Thr 157 . . . B T . . 1.18 −0.51 * . F 1.92 1.79 Ile 158 A . . B . . . 1.18 −0.09 . . F 1.08 1.23 Gln 159 . . . . T T . 0.93 −0.19 . . F 2.04 1.07 Lys 160 . . . . T T . 0.93 0.14 * . F 1.60 1.16 Gly 161 . . . . T T . 0.44 0.14 * . F 1.44 2.38 Ser 162 . . . . T T . −0.10 0.24 * . F 1.28 1.19 Tyr 163 . . . B T . . 0.58 0.49 * . . 0.12 0.44 Thr 164 . . B B . . . 0.29 0.91 * . . −0.44 0.69 Phe 165 . . B B . . . −0.57 1.40 * . . −0.60 0.54 Val 166 . . B B . . . −1.03 1.70 . . . −0.60 0.29 Pro 167 . . B B . . . −1.03 1.63 . . . −0.60 0.16 Trp 168 A . . B . . . −1.49 1.53 . * . −0.60 0.25 Leu 169 A . . B . . . −1.13 1.53 * . . −0.60 0.29 Leu 170 A . . B . . . −0.32 0.89 * . . −0.30 0.38 Ter 171 A . . . . . . 0.19 0.46 * . . 0.20 0.71 Phe 172 . . . . T . . 0.10 −0.03 * . . 1.80 0.85 Lys 173 . . . . T T . −0.20 −0.33 * . F 2.60 1.38 Arg 174 . . . . . T C −0.20 −0.51 . . F 3.00 1.04 Gly 175 . . . . . T C 0.61 −0.21 . . F 2.25 0.99 Ser 176 A . . . . T . 0.91 −1.00 * . F 2.05 0.86 Ala 177 A A . . . . . 1.66 −1.00 * . F 1.35 0.76 Leu 178 A A . . . . . 1.61 −1.00 . . F 1.20 1.54 Glu 179 A A . . . . . 1.50 −1.43 . . F 0.90 1.98 Glu 180 A A . . . . . 1.89 −1.41 * . F 0.90 3.16 Lys 181 A A . . . . . 1.30 −1.91 * . F 0.90 7.66 Glu 182 A A . . . . . 1.08 −1.91 . . F 0.90 3.10 Asn 183 A A . . . . . 1.03 −1.23 * * F 0.90 1.48 Lys 184 A A . . . . . 1.08 −0.59 * . F 0.75 0.55 Ile 185 A A . . . . . 1.08 −0.59 * * . 0.60 0.63 Leu 186 A A . . . . . 0.72 −0.59 * * . 0.60 0.68 Val 187 A A . . . . . 0.38 −0.50 . * . 0.30 0.49 Lys 188 A A . . . . . 0.13 −0.07 * * F 0.45 0.69 Glu 189 A . . . . T . −0.61 0.00 * * F 0.40 1.32 Thr 190 . . . . T T . −0.42 0.10 . * F 0.80 1.54 Gly 191 . . . . T T . −0.50 0.24 * . F 0.65 0.67 Tyr 192 . . . . T T . 0.11 0.93 * * . 0.20 0.27 Phe 193 . . B B . . . −0.28 1.69 . . . −0.60 0.29 Phe 194 . . B B . . . −0.28 1.63 . * . −0.60 0.29 Ile 195 . . B B . . . −0.82 1.60 . . . −0.60 0.32 Tyr 196 . . B B . . . −1.29 1.49 . . . −0.60 0.28 Gly 197 . . . B T . . −1.29 1.39 . . . −0.20 0.26 Gln 198 . . . B T . . −0.90 1.36 . . . −0.20 0.59 Val 199 . . . B . . C −0.20 1.16 . . . −0.40 0.54 Leu 200 . . . B . . C 0.73 0.40 . . . −0.10 0.92 Tyr 201 . . . . T T . 0.67 −0.03 . . . 1.25 1.06 Thr 202 . . . . T T . 0.77 0.06 . . F 0.80 2.06 Asp 203 . . . . T T . 0.18 0.17 . . F 0.80 3.91 Lys 204 A . . . . T . 0.43 −0.01 . . F 1.00 2.52 Thr 205 A A . . . . . 0.90 −0.16 . . F 0.60 1.73 Tyr 206 A A . . . . . 1.11 −0.21 . . . 0.45 1.03 Ala 207 A A . . . . . 0.61 0.29 . . . −0.30 0.70 Met 208 A A . . . . . −0.28 0.97 . . . −0.60 0.40 Gly 209 A A . B . . . −0.32 1.17 * . . −0.60 0.18 His 210 A A . B . . . 0.10 0.81 * . . −0.60 0.31 Leu 211 A A . B . . . 0.39 0.31 . . . −0.30 0.61 Ile 212 A A . B . . . 1.02 −0.30 . . . 0.45 1.22 Gln 213 A A . B . . . 0.77 −0.73 . * . 0.75 1.80 Arg 214 A A . B . . . 1.08 −0.59 . * F 0.90 1.62 Lys 215 A A . B . . . 0.26 −0.77 * * F 0.90 3.14 Lys 216 A A . B . . . 0.37 −0.81 . * F 0.90 1.35 Val 217 . A B B . . . 0.91 −0.43 * * . 0.30 0.60 His 218 . A B B . . . 0.91 −0.00 . * . 0.30 0.29 Val 219 . A B B . . . 0.80 −0.00 * * . 0.30 0.25 Phe 220 . . B B . . . −0.06 −0.00 * . . 0.30 0.57 Gly 221 A . . B . . . −0.40 0.04 . * . −0.30 0.35 Asp 222 A . . . . . . −0.36 −0.07 * . . 0.50 0.63 Glu 223 A . . . . . . −1.18 −0.03 * . . 0.50 0.60 Leu 224 A . . B . . . −0.63 −0.17 . . . 0.30 0.45 Ser 225 A . . B . . . −0.74 −0.11 . . . 0.30 0.39 Leu 226 A . . B . . . −1.10 0.57 . * . −0.60 0.18 Val 227 A . . B . . . −0.99 1.36 . * . −0.60 0.19 Thr 228 A . . B . . . −1.66 0.67 * * . −0.60 0.28 Leu 229 A . . B . . . −1.73 0.86 * . . −0.60 0.18 Phe 230 A . . B . . . −1.43 0.86 * . . −0.60 0.17 Arg 231 A . . B . . . −0.62 0.61 * . . −0.60 0.21 Cys 232 . . . B T . . −0.37 0.53 * . . −0.20 0.41 Ile 233 . . . B T . . −0.27 0.46 * . . −0.20 0.46 Gln 234 . . . B T . . 0.54 0.10 * . . 0.10 0.37 Asn 235 . . . B . . C 0.93 0.10 * . . 0.05 1.19 Met 236 . . . B . . C 0.01 0.01 * . F 0.20 2.44 Pro 237 . . . B . . C 0.47 0.01 * . F 0.44 1.16 Glu 238 . . . . T . . 1.36 0.04 * . F 1.08 1.12 Thr 239 . . . . . . C 1.36 0.04 * . F 1.12 1.82 Leu 240 . . . . . . C 1.06 −0.17 * . F 1.96 1.89 Pro 241 . . . . T . . 0.99 −0.21 . . F 2.40 1.46 Asn 242 . . . . T . . 0.96 0.36 . . F 1.41 0.54 Asn 243 . . . . T T . 0.66 0.63 . . F 1.22 1.03 Ser 244 . . . . T T . 0.38 0.33 . . F 1.13 0.89 Cys 245 . . . . T T . 0.84 0.40 . . . 0.74 0.56 Tyr 246 . . . . T T . 0.17 0.43 . . . 0.20 0.35 Ser 247 A . . . . . . −0.42 0.71 . . . −0.40 0.18 Ala 248 A A . . . . . −0.38 0.83 . . . −0.60 0.34 Gly 249 A A . . . . . −0.89 0.26 . . . −0.30 0.43 Ile 250 A A . . . . . −0.22 0.19 * . . −0.30 0.27 Ala 251 A A . . . . . 0.02 −0.20 * . . 0.30 0.46 Lys 252 A A . . . . . −0.02 −0.70 . . . 0.60 0.80 Leu 253 A A . . . . . 0.57 −0.70 . . F 0.90 1.13 Glu 254 A A . . . . . 0.91 −1.39 . . F 0.90 1.87 Glu 255 A A . . . . . 0.99 −1.89 . . F 0.90 1.62 Gly 256 A A . . . . . 1.58 −1.20 . * F 0.90 1.62 Asp 257 A A . . . . . 0.72 −1.49 . * F 0.90 1.62 Glu 258 A A . . . . . 0.94 −0.80 * * F 0.75 0.77 Leu 259 A A . . . . . 0.06 −0.30 * * . 0.30 0.79 Gln 260 A A . . . . . −0.16 −0.04 * . . 0.30 0.33 Leu 261 A A . . . . . 0.30 0.39 * . . −0.30 0.30 Ala 262 A A . . . . . 0.30 0.39 * . . −0.30 0.70 Ile 263 A A . . . . . 0.30 −0.30 . * . 0.30 0.70 Pro 264 A . . . . T . 0.52 −0.30 . * F 1.00 1.37 Arg 265 A . . . . T . 0.52 −0.49 . * F 1.00 1.37 Glu 266 A . . . . T . 0.44 −0.59 * * F 1.30 3.38 Asn 267 A . . . . T . 0.73 −0.59 * * F 1.30 1.53 Ala 268 A . . . . . . 0.81 −0.63 * * . 0.95 1.05 Gln 269 A . . . . . . 1.02 0.06 * * . −0.10 0.50 Ile 270 A . . . . . . 0.57 0.06 . * . 0.15 0.52 Ser 271 . . . . . . C 0.57 0.09 . * . 0.60 0.51 Leu 272 . . . . . . C −0.29 −0.41 . * F 1.60 0.49 Asp 273 . . . . T T . −0.01 −0.17 . * F 2.25 0.52 Gly 274 . . . . T T . −0.71 −0.37 . * F 2.50 0.56 Asp 275 . . . . T T . −0.52 0.03 . * F 1.65 0.59 Val 276 A . . . . T . −0.57 0.13 . * F 1.00 0.30 Thr 277 A . . B . . . −0.34 0.56 . * . −0.10 0.30 Phe 278 A . . B . . . −1.16 0.63 . * . −0.35 0.18 Phe 279 A . . B . . . −0.77 1.31 . * . −0.60 0.20 Gly 280 A A . . . . . −1.58 0.67 . * . −0.60 0.28 Ala 281 A A . . . . . −1.53 0.87 . * . −0.60 0.27 Leu 282 A A . . . . . −1.61 0.77 * . . −0.60 0.26 Lys 283 A A . . . . . −1.30 0.41 * . . −0.60 0.33 Leu 284 A A . . . . . −0.99 0.41 . . . −0.60 0.42 Leu 285 A A . . . . . −1.03 0.34 * . . −0.30 0.65

TABLE 10 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A . . . . . . 0.73 −0.71 . . . 0.95 1.39 Asp 2 A . . . . T . 1.12 −0.66 * . . 1.15 1.56 Asp 3 A . . . . T . 1.62 −1.09 * . . 1.15 2.12 Ser 4 A . . . . T . 2.01 −1.51 . . . 1.15 4.19 Thr 5 A . . . . T . 2.40 −2.13 . . F 1.30 4.35 Glu 6 A A . . . . . 2.70 −1.73 * * F 0.90 4.51 Arg 7 A A . . . . . 2.81 −1.34 * * F 0.90 4.51 Glu 8 A A . . . . . 2.00 −1.73 * * F 0.90 6.12 Gln 9 A A . . . . . 1.99 −1.53 * * F 0.90 2.91 Ser 10 A . . B . . . 2.00 −1.04 * * F 0.90 2.15 Arg 11 A . . B . . . 1.33 −0.66 * * F 0.90 1.66 Leu 12 A . . B . . . 0.41 −0.09 * * F 0.45 0.51 Thr 13 A . . B . . . 0.46 0.20 * * F −0.15 0.32 Ser 14 A A . . . . . 0.50 −0.19 * * . 0.30 0.32 Cys 15 A A . . . . . 0.91 −0.19 * * . 0.30 0.78 Leu 16 A A . . . . . 0.80 −0.87 * * F 0.90 1.06 Lys 17 A A . . . . . 1.61 −1.36 . * F 0.90 1.37 Lys 18 A A . . . . . 1.32 −1.74 . * F 0.90 4.44 Arg 19 A A . . . . . 1.67 −1.70 . * F 0.90 5.33 Glu 20 A A . . . . . 1.52 −2.39 . * F 0.90 5.33 Glu 21 A A . . . . . 2.38 −1.70 . * F 0.90 2.20 Met 22 A A . . . . . 2.33 −1.70 . * F 0.90 2.24 Lys 23 A A . . . . . 1.62 −1.70 * * F 0.90 2.24 Leu 24 A A . . . . . 0.66 −1.13 * * F 0.75 0.69 Lys 25 A A . . . . . 0.36 −0.49 . * F 0.45 0.52 Glu 26 A A . B . . . −0.53 −0.71 * * . 0.60 0.35 Cys 27 A A . B . . . −0.74 −0.03 * * . 0.30 0.30 Val 28 A A . B . . . −1.00 −0.03 * * . 0.30 0.12 Ser 29 A A . B . . . −0.08 0.40 * * . −0.30 0.11 Ile 30 A . . B . . . −0.08 0.40 * * . −0.30 0.40 Leu 31 A . . B . . . −0.08 −0.17 * . . 0.45 1.08 Pro 32 . . . B . . C 0.29 −0.81 * . F 1.10 1.39 Arg 33 . . . . T . . 0.93 −0.81 . * F 1.50 2.66 Lys 34 . . . . T . . 0.93 −1.07 . . F 1.84 4.98 Glu 35 . . . . . . C 0.97 −1.37 * * F 1.98 4.32 Ser 36 . . . . . T C 1.89 −1.16 * * F 2.52 1.64 Pro 37 . . . . . T C 1.80 −1.16 * * F 2.86 1.60 Ser 38 . . . . T T . 1.39 −0.77 * . F 3.40 1.24 Val 39 A . . . . T . 1.39 −0.39 . * F 2.36 1.24 Arg 40 A . . . . . . 1.39 −0.77 * * F 2.46 1.60 Ser 41 A . . . . . . 1.34 −1.20 * * F 2.46 2.00 Ser 42 . . . . T T . 1.60 −1.16 . * F 3.06 2.67 Lys 43 . . . . T T . 1.09 −1.80 * * F 3.06 2.72 Asp 44 . . . . T T . 1.13 −1.11 * * F 3.40 1.67 Gly 45 A . . . . T . 0.43 −0.81 * * F 2.66 1.03 Lys 46 A A . . . . . 0.14 −0.70 . . F 1.77 0.52 Leu 47 A A . . . . . 0.13 −0.20 * . . 0.98 0.31 Leu 48 A A . . . . . −0.72 0.29 * . . 0.04 0.46 Ala 49 A A . . . . . −1.53 0.54 . * . −0.60 0.19 Ala 50 A A . . . . . −2.00 1.23 . . . −0.60 0.19 Thr 51 A A . . . . . −2.63 1.23 . . . −0.60 0.19 Leu 52 A A . . . . . −2.63 1.04 . . . −0.60 0.19 Leu 53 A A . . . . . −2.63 1.23 . . . −0.60 0.15 Leu 54 A A . . . . . −2.34 1.41 . . . −0.60 0.09 Ala 55 A A . . . . . −2.42 1.31 . . . −0.60 0.14 Leu 56 A A . . . . . −2.78 1.20 . . . −0.60 0.09 Leu 57 A . . . . T . −2.78 1.09 . . . −0.20 0.06 Ser 58 A . . . . T . −2.28 1.09 . . . −0.20 0.05 Cys 59 A . . . . T . −2.32 1.07 . . . −0.20 0.09 Cys 60 A . . . . T . −2.59 1.03 . . . −0.20 0.08 Leu 61 . . B B . . . −2.08 0.99 . . . −0.60 0.04 Thr 62 . . B B . . . −1.97 0.99 . . . −0.60 0.11 Val 63 . . B B . . . −1.91 1.20 . . . −0.60 0.17 Val 64 . . B B . . . −1.24 1.39 . . . −0.60 0.33 Ser 65 . . B B . . . −1.43 1.10 . . . −0.60 0.40 Phe 66 A . . B . . . −1.21 1.26 . . . −0.60 0.40 Tyr 67 A . . B . . . −1.49 1.11 . . . −0.60 0.54 Gln 68 A . . B . . . −1.44 0.97 . . . −0.60 0.41 Val 69 A . . B . . . −0.59 1.27 . . . −0.60 0.39 Ala 70 A . . B . . . −0.63 0.89 . . . −0.60 0.43 Ala 71 A . . B . . . 0.07 0.56 . * . −0.60 0.25 Leu 72 A . . . . T . −0.50 0.16 . . . 0.10 0.55 Gln 73 A . . . . T . −1.09 0.20 . . F 0.25 0.45 Gly 74 A . . . . T . −0.53 0.20 . . F 0.25 0.45 Asp 75 A . . . . T . −0.76 0.09 . * F 0.25 0.73 Leu 76 A A . . . . . −0.06 0.09 . * F −0.15 0.35 Ala 77 A A . . . . . 0.17 −0.31 . * . 0.30 0.69 Ser 78 A A . . . . . 0.17 −0.24 . * . 0.30 0.42 Leu 79 A A . . . . . −0.30 −0.24 . * . 0.30 0.88 Arg 80 A A . . . . . −0.30 −0.24 . * . 0.30 0.72 Ala 81 A A . . . . . 0.17 −0.34 . * . 0.30 0.93 Glu 82 A A . . . . . 0.72 −0.30 . * . 0.45 1.11 Leu 83 A A . . . . . 0.99 −0.49 . * . 0.30 0.77 Gln 84 A A . . . . . 1.21 0.01 . * . −0.15 1.04 Gly 85 A A . . . . . 1.10 0.01 * * . −0.30 0.61 His 86 A A . . . . . 1.73 0.01 * * . −0.15 1.27 His 87 A A . . . . . 0.92 −0.67 . * . 0.75 1.47 Ala 88 A A . . . . . 1.52 −0.39 . * . 0.45 1.22 Glu 89 A A . . . . . 0.93 −0.39 . . . 0.45 1.39 Lys 90 A A . . . . . 0.93 −0.39 * . F 0.60 1.03 Leu 91 A . . . . T . 0.38 −0.46 * . . 0.85 1.01 Pro 92 A . . . . T . 0.07 −0.46 . . . 0.70 0.59 Ala 93 A . . . . T . 0.07 −0.03 . . . 0.70 0.29 Gly 94 A . . . . T . −0.14 0.47 . . . −0.20 0.36 Ala 95 A . . . . . . −0.14 0.21 . * . −0.10 0.36 Gly 96 A . . . . . . 0.08 −0.21 . . F 0.65 0.71 Ala 97 A . . . . . . −0.06 −0.21 . . F 0.65 0.72 Pro 98 A . . . . . . −0.28 −0.21 . * F 0.65 0.71 Lys 99 A A . . . . . 0.07 −0.03 . . F 0.45 0.59 Ala 100 A A . . . . . 0.66 −0.46 . . F 0.60 1.01 Gly 101 A A . . . . . 0.41 −0.96 . . F 0.90 1.13 Leu 102 A A . . . . . 0.79 −0.89 . . F 0.75 0.57 Glu 103 A A . . . . . 0.41 −0.46 * . F 0.45 0.88 Glu 104 A A . . . . . −0.49 −0.46 * . F 0.45 0.89 Ala 105 A A . . . . . −0.21 −0.24 . . . 0.30 0.81 Pro 106 A A . . . . . −0.46 −0.44 . . . 0.30 0.67 Ala 107 A A . . . . . 0.01 0.06 . . . −0.30 0.39 Val 108 A A . . . . . −0.80 0.49 * * . −0.60 0.38 Thr 109 A A . . . . . −0.76 0.67 . * . −0.60 0.20 Ala 110 A A . . . . . −1.06 0.24 * * . −0.30 0.40 Gly 111 A A . . . . . −1.54 0.43 * * . −0.60 0.38 Leu 112 A A . . . . . −0.96 0.57 * * . −0.60 0.23 Lys 113 . A B . . . . −0.31 0.09 * * . −0.30 0.39 Ile 114 . A B . . . . −0.21 0.01 * . . −0.30 0.61 Phe 115 . A B . . . . −0.21 0.01 * . . 0.15 1.15 Glu 116 . A . . . . C −0.08 −0.17 * . F 1.25 0.58 Pro 117 . A . . . . C 0.39 0.26 * * F 1.10 1.28 Pro 118 . . . . . . C 0.34 0.00 * . F 2.20 1.47 Ala 119 . . . . . T C 0.89 −0.79 . * F 3.00 1.47 Pro 120 . . . . . T C 1.59 −0.36 . * F 2.25 0.94 Gly 121 . . . . T T . 1.29 −0.39 . * F 2.15 0.98 Glu 122 . . . . T T . 1.20 −0.43 . . F 2.00 1.30 Gly 123 . . . . . . C 1.41 −0.54 . . F 1.60 1.12 Asn 124 . . . . . T C 2.00 −0.57 . . F 1.50 1.97 Ser 125 . . . . . T C 1.91 −0.60 . * F 1.50 1.82 Ser 126 . . . . . T C 2.37 −0.21 . * F 1.54 2.47 Gln 127 . . . . . T C 2.37 −0.64 . * F 2.18 3.01 Asn 128 . . . . . . C 2.76 −0.64 . . F 2.32 3.61 Ser 129 . . . . . T C 2.87 −1.03 . . F 2.86 5.39 Arg 130 . . . . T T . 2.58 −1.41 * . F 3.40 6.09 Asn 131 . . . . T T . 2.02 −1.31 * . F 3.06 3.83 Lys 132 . . . . T T . 2.02 −1.07 * . F 2.72 2.12 Arg 133 . . . . T . . 1.68 −1.06 * . F 2.18 1.88 Ala 134 . . . . . . C 1.77 −0.63 * . F 1.64 1.15 Val 135 . . . . . . C 1.66 −0.60 * . F 1.15 0.89 Gln 136 . . . . . . C 1.66 −0.60 * . F 1.49 0.79 Gly 137 . . . . . T C 1.30 −0.60 * . F 2.18 1.35 Pro 138 . . . . . T C 0.84 −0.61 * . F 2.52 2.63 Glu 139 . . . . . T C 1.13 −0.83 * . F 2.86 1.50 Glu 140 . . . . T T . 1.74 −0.84 . . F 3.40 2.03 Thr 141 . . . . T . . 1.43 −0.51 . . F 2.86 2.06 Gly 142 . . . . T T . 1.08 −0.46 . . F 2.42 1.72 Ser 143 . . . . T T . 0.43 0.33 . . F 1.33 0.86 Tyr 144 . . . . T T . 0.22 0.97 . . . 0.54 0.44 Thr 145 . . . . T T . −0.07 0.91 . . . 0.20 0.69 Phe 146 . . B B . . . −0.57 1.40 . . . −0.60 0.54 Val 147 . . B B . . . −1.03 1.70 . . . −0.60 0.29 Pro 148 . . B B . . . −1.03 1.63 . . . −0.60 0.16 Trp 149 A . . B . . . −1.49 1.53 . * . −0.60 0.25 Leu 150 A . . B . . . −1.13 1.53 * . . −0.60 0.29 Leu 151 A . . B . . . −0.32 0.89 * . . −0.30 0.38 Ser 152 A . . . . . . 0.19 0.46 * . . 0.20 0.71 Phe 153 . . . . T . . 0.10 −0.03 * . . 1.80 0.85 Lys 154 . . . . T T . −0.20 −0.33 * . F 2.60 1.38 Arg 155 . . . . . T C −0.20 −0.51 . . F 3.00 1.04 Gly 156 . . . . . T C 0.61 −0.21 . . F 2.25 0.99 Ser 157 A . . . . T . 0.91 −1.00 * . F 2.05 0.86 Ala 158 A A . . . . . 1.66 −1.00 * . F 1.35 0.76 Leu 159 A A . . . . . 1.61 −1.00 . . F 1.20 1.54 Glu 160 A A . . . . . 1.50 −1.43 . . F 0.90 1.98 Glu 161 A A . . . . . 1.89 −1.41 * . F 0.90 3.16 Lys 162 A A . . . . . 1.30 −1.91 * . F 0.90 7.66 Glu 163 A A . . . . . 1.08 −1.91 . . F 0.90 3.10 Asn 164 A A . . . . . 1.03 −1.23 * * F 0.90 1.48 Lys 165 A A . . . . . 1.08 −0.59 * . F 0.75 0.55 Ile 166 A A . . . . . 1.08 −0.59 * * . 0.60 0.63 Leu 167 A A . . . . . 0.72 −0.59 * * . 0.76 0.68 Val 168 A A . . . . . 0.38 −0.50 . * . 0.92 0.49 Lys 169 A A . . . . . 0.13 −0.07 * * F 0.93 0.69 Glu 170 A . . . . T . −0.61 0.00 * * F 1.64 1.32 Thr 171 . . . . T T . −0.42 0.10 . * F 1.60 1.54 Gly 172 . . . . T T . −0.50 0.24 * . F 1.29 0.67 Tyr 173 . . . . T T . 0.11 0.93 * * . 0.68 0.27 Phe 174 . . B B . . . −0.28 1.69 . . . −0.28 0.29 Phe 175 . . B B . . . −0.28 1.63 . * . −0.44 0.29 Ile 176 . . B B . . . −0.82 1.60 . . . −0.60 0.32 Tyr 177 . . B B . . . −1.29 1.49 . . . −0.60 0.28 Gly 178 . . . B T . . −1.29 1.39 . . . −0.20 0.26 Gln 179 . . . B T . . −0.90 1.36 . . . −0.20 0.59 Val 180 . . . B . . C −0.20 1.16 . . . −0.40 0.54 Leu 181 . . . B . . C 0.73 0.40 . . . −0.10 0.92 Tyr 182 . . . . T T . 0.67 −0.03 . . . 1.25 1.06 Thr 183 . . . . T T . 0.77 0.06 . . F 0.80 2.06 Asp 184 . . . . T T . 0.18 0.17 . . F 0.80 3.91 Lys 185 A . . . . T . 0.43 −0.01 . . F 1.00 2.52 Thr 186 A A . . . . . 0.90 −0.16 . . F 0.60 1.73 Tyr 187 A A . . . . . 1.11 −0.21 . . . 0.45 1.03 Ala 188 A A . . . . . 0.61 0.29 . . . −0.30 0.70 Met 189 A A . . . . . −0.28 0.97 . . . −0.60 0.40 Gly 190 A A . B . . . −0.32 1.17 * . . −0.60 0.18 His 191 A A . B . . . 0.10 0.81 * . . −0.60 0.31 Leu 192 A A . B . . . 0.39 0.31 . . . −0.30 0.61 Ile 193 A A . B . . . 1.02 −0.30 . . . 0.45 1.22 Gln 194 A A . B . . . 0.77 −0.73 . * . 0.75 1.80 Arg 195 A A . B . . . 1.08 −0.59 * * F 0.90 1.62 Lys 196 A A . B . . . 0.26 −0.77 * * F 0.90 3.14 Lys 197 A A . B . . . 0.37 −0.81 . * F 0.90 1.35 Val 198 . A B B . . . 0.91 −0.43 * * . 0.30 0.60 His 199 . A B B . . . 0.91 0.00 * * . 0.30 0.29 Val 200 . A B B . . . 0.80 0.00 * * . 0.30 0.25 Phe 201 . . B B . . . −0.06 0.00 * . . 0.30 0.57 Gly 202 A . . B . . . −0.40 0.04 . * . −0.30 0.35 Asp 203 A . . . . . . −0.36 −0.07 * . . 0.50 0.63 Glu 204 A . . . . . . −1.18 −0.03 * . . 0.50 0.60 Leu 205 A . . B . . . −0.63 −0.17 . . . 0.30 0.45 Ser 206 A . . B . . . −0.74 −0.11 . . . 0.30 0.39 Leu 207 A . . B . . . −1.10 0.57 . * . −0.60 0.18 Val 208 A . . B . . . −0.99 1.36 . * . −0.60 0.19 Thr 209 A . . B . . . −1.66 0.67 * * . −0.60 0.28 Leu 210 A . . B . . . −1.73 0.86 * . . −0.60 0.18 Phe 211 A . . B . . . −1.43 0.86 * . . −0.60 0.17 Arg 212 A . . B . . . −0.62 0.61 * . . −0.60 0.21 Cys 213 . . . B T . . −0.37 0.53 * . . −0.20 0.41 Ile 214 . . . B T . . −0.27 0.46 * . . −0.20 0.46 Gln 215 . . . B T . . 0.54 0.10 * . . 0.10 0.37 Asn 216 . . . B . . C 0.93 0.10 * . . 0.05 1.19 Met 217 . . . B . . C 0.01 0.01 * . F 0.20 2.44 Pro 218 . . . B . . C 0.47 0.01 * . F 0.44 1.16 Glu 219 . . . . T . . 1.36 0.04 * . F 1.08 1.12 Thr 220 . . . . . . C 1.36 0.04 * . F 1.12 1.82 Leu 221 . . . . . . C 1.06 −0.17 * . F 1.96 1.89 Pro 222 . . . . T . . 0.99 −0.21 . . F 2.40 1.46 Asn 223 . . . . T . . 0.96 0.36 . . F 1.41 0.54 Asn 224 . . . . T T . 0.66 0.63 . . F 1.22 1.03 Ser 225 . . . . T T . 0.38 0.33 . . F 1.13 0.89 Cys 226 . . . . T T . 0.84 0.40 . . . 0.74 0.56 Tyr 227 . . . . T T . 0.17 0.43 . . . 0.20 0.35 Ser 228 A . . . . . . −0.42 0.71 . . . −0.40 0.18 Ala 229 A A . . . . . −0.38 0.83 . . . −0.60 0.34 Gly 230 A A . . . . . −0.89 0.26 . . . −0.30 0.43 Ile 231 A A . . . . . −0.22 0.19 * . . −0.30 0.27 Ala 232 A A . . . . . 0.02 −0.20 * . . 0.30 0.46 Lys 233 A A . . . . . −0.02 −0.70 . . . 0.60 0.80 Leu 234 A A . . . . . 0.57 −0.70 . . F 0.90 1.13 Glu 235 A A . . . . . 0.91 −1.39 . . F 0.90 1.87 Glu 236 A A . . . . . 0.99 −1.89 . . F 0.90 1.62 Gly 237 A A . . . . . 1.58 −1.20 . * F 0.90 1.62 Asp 238 A A . . . . . 0.72 −1.49 . * F 0.90 1.62 Glu 239 A A . . . . . 0.94 −0.80 * * F 0.75 0.77 Leu 240 A A . . . . . 0.06 −0.30 * * . 0.30 0.79 Gln 241 A A . . . . . −0.16 −0.04 * . . 0.30 0.33 Leu 242 A A . . . . . 0.30 0.39 * . . −0.30 0.30 Ala 243 A A . . . . . 0.30 0.39 * . . −0.30 0.70 Ile 244 A A . . . . . 0.30 −0.30 . * . 0.30 0.70 Pro 245 A . . . . T . 0.52 −0.30 . * F 1.00 1.37 Arg 246 A . . . . T . 0.52 −0.49 . * F 1.00 1.37 Glu 247 A . . . . T . 0.44 −0.59 * * F 1.30 3.38 Asn 248 A . . . . T . 0.73 −0.59 * * F 1.30 1.53 Ala 249 A . . . . . . 0.81 −0.63 * * . 0.95 1.05 Gln 250 A . . . . . . 1.02 0.06 * * . −0.10 0.50 Ile 251 A . . . . . . 0.57 0.06 * * . 0.15 0.52 Ser 252 . . . . . . C 0.57 0.09 . * . 0.60 0.51 Leu 253 . . . . . . C −0.29 −0.41 . * F 1.60 0.49 Asp 254 . . . . T T . −0.01 −0.17 . * F 2.25 0.52 Gly 255 . . . . T T . −0.71 −0.37 . * F 2.50 0.56 Asp 256 . . . . T T . −0.52 0.03 . * F 1.65 0.59 Val 257 A . . . . T . −0.57 0.13 . * F 1.00 0.30 Thr 258 A . . B . . . −0.34 0.56 . * . −0.10 0.30 Phe 259 A . . B . . . −1.16 0.63 . * . −0.35 0.18 Phe 260 A . . B . . . −0.77 1.31 . * . −0.60 0.20 Gly 261 A A . . . . . −1.58 0.67 . * . −0.60 0.28 Ala 262 A A . . . . . −1.53 0.87 . * . −0.60 0.27 Leu 263 A A . . . . . −1.61 0.77 * . . −0.60 0.26 Lys 264 A A . . . . . −1.30 0.41 * . . −0.60 0.33 Leu 265 A A . . . . . −0.99 0.41 . . . −0.60 0.42 Leu 266 A A . . . . . −1.03 0.34 * . . −0.30 0.65

In another embodiment, the invention provides antibodies that bind a polypeptide comprising, or alternatively consisting of, an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion may be an immunogenic or antigenic epitope of a polypeptide of the invention. An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

As to the selection of polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) “Antibodies that react with predetermined sites on proteins”, Science, 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.

In specific embodiments, antibodies of the present invention bind antigenic epitope-bearing peptides and polypeptides of B Lymphocyte Stimulator and preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids contained within the amino acid sequence of a B Lymphocyte Stimulator polypeptide. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate B Lymphocyte Stimulator-specific antibodies and which may be bound by the antibodies of the invention include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Phe-115 to about Leu-147 in SEQ ID NO:3228; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ile-150 to about Tyr-163 in SEQ ID NO:3228; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-171 to about Phe-194 in SEQ ID NO:3228; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Glu-223 to about Tyr-246 in SEQ ID NO:3228; and a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-271 to about Phe-278 in FIGS. 1A and 1B (SEQ ID NO:3228). In this context, “about” means the particularly recited ranges and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid residues at either or both the amino- and carboxy-termini. These polypeptide fragments have been determined to bear antigenic epitopes of the B Lymphocyte Stimulator polypeptide by the analysis of the Jameson-Wolf antigenic index, as disclosed Table 9, above.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate B Lymphocyte Stimulator-specific antibodies and which may be bound by the antibodies of the invention include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Pro-32 to about Leu-47 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Glu-116 to about Ser-143 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Phe-153 to about Tyr-173 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Pro-218 to about Tyr-227 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ala-232 to about Gln-241 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ile-244 to about Ala-249 in SEQ ID NO:3229; and a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-252 to about Val-257 in SEQ ID NO:3229. In this context, “about” means the particularly recited ranges and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid residues at either or both the amino- and carboxy-termini. These polypeptide fragments have been determined to bear antigenic epitopes of the B Lymphocyte Stimulator polypeptide by the analysis of the Jameson-Wolf antigenic index, as disclosed in Table 10 generated by the Protean component of the DNA*STAR computer program (as set forth above).

B Lymphocyte Stimulator epitope-bearing peptides and polypeptides may be produced by any conventional means. See, e.g., Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135; this “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

The present invention encompasses antibodies that bind polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:3228, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC™ deposit No. 97768, or encoded by a polynucleotide that hybridizes to cDNA sequence contained in ATCC™ deposit No. 97768 (e.g., under hybridization conditions described herein).

The present invention also encompasses antibodies that bind polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:3229, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC™ deposit No. 203518, or encoded by a polynucleotide that hybridizes to the cDNA sequence contained in ATCC™ deposit No. 203518 (e.g., under hybridization conditions described herein).

The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses antibodies that bind a polypeptide comprising an epitope. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

B Lymphocyte Stimulator polypeptide fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antibodies of the present invention bind antigenic epitopes preferably containing a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes that may be bound by antibodies of the present invention are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes of B Lymphocyte Stimulator may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

Epitope-bearing B Lymphocyte Stimulator polypeptides may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, the antibodies of the present invention may bind polypeptides comprising an immunogenic or antigenic epitope fused to other polypeptide sequences. For example, the B Lymphocyte Stimulator polypeptides may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof), or albumin (including but not limited to recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988) Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

In another embodiment, the antibodies of the present invention bind B Lymphocyte Stimulator polypeptides and/or the epitope-bearing fragments thereof that are fused with a heterologous antigen (e.g., polypeptide, carbohydrate, phospholipid, or nucleic acid). In specific embodiments, the heterologous antigen is an immunogen.

In a more specific embodiment, the heterologous antigen is the gp120 protein of HIV, or a fragment thereof.

In another embodiment, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides and/or the epitope-bearing fragments thereof that are fused with polypeptide sequences of another TNF ligand family member (or biologically active fragments or variants thereof). In a specific embodiment, the antibodies of the present invention bind B Lymphocyte Stimulator polypeptides of the present invention are fused with a CD40L polypeptide sequence. In a preferred embodiment, the CD40L polypeptide sequence is soluble.

In another embodiment, antibodies of the present invention bind mutant B Lymphocyte Stimulator polypeptides that have been generated by random mutagenesis of a polynucleotide encoding the B Lymphocyte Stimulator polypeptide, by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, antibodies of the present invention bind one or more components, motifs, sections, parts, domains, fragments, etc., of B Lymphocyte Stimulator recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are, for example, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha (International Publication No. WO 98/07880), OPG, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), TR12, CAD, and v-FLIP. In further embodiments, the heterologous molecules are any member of the TNF family.

In another preferred embodiment, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides of the invention (including biologically active fragments or variants thereof), that are fused with soluble APRIL polypeptides (e.g., amino acid residues 105 through 250 of SEQ ID NO:3239), or biologically active fragments or variants thereof.

To improve or alter the characteristics of B Lymphocyte Stimulator polypeptides, protein engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or “muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. For instance, for many proteins, including the extracellular domain or the mature form(s) of a secreted protein, it is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function. For instance, Ron et al., J. Biol. Chem., 268:2984-2988 (1993) reported modified KGF proteins that had heparin binding activity even if 3, 8, or 27 amino-terminal amino acid residues were missing. Accordingly, antibodies of the present invention may bind B Lymphocyte Stimulator polypeptide mutants or variants generated by protein engineering.

In the present case, since the protein of the invention is a member of the TNF polypeptide family, deletions of N-terminal amino acids up to the Gly (G) residue at position 191 in SEQ ID NO:3228 may retain some biological activity such as, for example, the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and cytotoxicity to appropriate target cells. Polypeptides having further N-terminal deletions including the Gly (G) residue would not be expected to retain biological activities because it is known that this residue in TNF-related polypeptides is in the beginning of the conserved domain required for biological activities. However, even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities may still be retained. Thus, the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or extracellular domain of the protein generally will be retained when less than the majority of the residues of the complete or extracellular domain of the protein are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the B Lymphocyte Stimulator of SEQ ID NO:3228, up to the glycine residue at position 191 (Gly-191 residue from the amino terminus). In particular, the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n¹-285 of SEQ ID NO:3228, where n¹ is an integer in the range of the amino acid position of amino acid residues 2-190 of the amino acid sequence in SEQ ID NO:3228. More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues 2-285, 3-285, 4-285, 5-285, 6-285, 7-285, 8-285, 9-285, 10-285, 11-285, 12-285, 13-285, 14-285, 15-285, 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-285, 71-285, 72-285, 73-285, 74-285, 75-285, 76-285, 77-285, 78-285, 79-285, 80-285, 81-285, 82-285, 83-285, 84-285, 85-285, 86-285, 87-285, 88-285, 89-285, 90-285, 91-285, 92-285, 93-285, 94-285, 95-285, 96-285, 97-285, 98-285, 99-285, 100-285, 101-285, 102-285, 103-285, 104-285, 105-285, 106-285, 107-285, 108-285, 109-285, 110-285, 111-285, 112-285, 113-285, 114-285, 115-285, 116-285, 117-285, 118-285, 119-285, 120-285, 121-285, 122-285, 123-285, 124-285, 125-285, 126-285, 127-285, 128-285, 129-285, 130-285, 131-285, 132-285, 133-285, 134-285, 135-285, 136-285, 137-285, 138-285, 139-285, 140-285, 141-285, 142-285, 143-285, 144-285, 145-285, 146-285, 147-285, 148-285, 149-285, 150-285, 151-285, 152-285, 153-285, 154-285, 155-285, 156-285, 157-285, 158-285, 159-285, 160-285, 161-285, 162-285, 163-285, 164-285, 165-285, 166-285, 167-285, 168-285, 169-285, 170-285, 171-285, 172-285, 173-285, 174-285, 175-285, 176-285, 177-285, 178-285, 179-285, 180-285, 181-285, 182-285, 183-285, 184-285, 185-285, 186-285, 187-285, 188-285, 189-285, and 190-285 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

Furthermore, since the predicted extracellular domain of the B Lymphocyte Stimulator polypeptides of the invention may itself elicit biological activity, deletions of N- and C-terminal amino acid residues from the predicted extracellular region of the polypeptide (spanning positions Gln-73 to Leu-285 of SEQ ID NO:3228) may retain some biological activity such as, for example, ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and modulation of cell replication or modulation of target cell activities. However, even if deletion of one or more amino acids from the N-terminus of the predicted extracellular domain of a B Lymphocyte Stimulator polypeptide results in modification or loss of one or more biological functions of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptides to induce and/or bind to antibodies which recognize the complete or mature or extracellular domains of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature or extracellular domains of the polypeptides are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of B Lymphocyte Stimulator shown in SEQ ID NO:3228, up to the glycine residue at position number 280. In particular, the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n²-285 of SEQ ID NO:3228, where n² is an integer in the range of the amino acid position of amino acid residues 73-280 in SEQ ID NO:3228, and 73 is the position of the first residue from the N-terminus of the predicted extracellular domain of the B Lymphocyte Stimulator polypeptide (disclosed in SEQ ID NO:3228). More in particular, in certain embodiments, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285; and G-280 to L-285 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

Highly preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence least 80%, 85%, 90% identical and more preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.

Preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 90% identical to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. More preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 95% identical to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. More preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 96% identical to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.

Additionally, more preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 97% to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. Additionally, more preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 98% to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. Additionally, more preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 99% identical to B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.

In specific embodiments, antibodies of the present invention bind polypeptides comprising, or alternatively consisting of, one of the following N-terminally deleted polypeptide fragments of B Lymphocyte Stimulator amino acid residues Ala-71 through Leu-285, amino acid residues Ala-81 through Leu-285, amino acid residues Leu-112 through Leu-285, amino acid residues Ala-134 through Leu-285, amino acid residues Leu-147 through Leu-285, and amino acid residues Gly-161 through Leu-285 of SEQ ID NO:3228.

Similarly, many examples of biologically functional C-terminal deletion polypeptides are known. For instance, Interferon gamma shows up to ten times higher activities by deleting 8-10 amino acid residues from the carboxy terminus of the protein (Döbeli et al., J. Biotechnology 7:199-216 (1988). Since the present protein is a member of the TNF polypeptide family, deletions of C-terminal amino acids up to the leucine residue at position 284 are expected to retain most if not all biological activity such as, for example, ligand binding, the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and modulation of cell replication. Polypeptides having deletions of up to about 10 additional C-terminal residues (i.e., up to the glycine residue at position 274) also may retain some activity such as receptor binding, although such polypeptides would lack a portion of the conserved TNF domain which extends to about Leu-284 of SEQ ID NO:3228. However, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities may still be retained. Thus, the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature protein generally will be retained when less than the majority of the residues of the complete or mature protein are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3228, up to the glycine residue at position 274 (Gly-274). In particular, the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m¹ of the amino acid sequence in SEQ ID NO:3228, where m¹ is any integer in the range of the amino acid position of amino acid residues 274-284 in SEQ ID NO:3228. More in particular, the invention provides antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues 1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1-282, 1-283 and 1-284 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

Also provided are antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively consisting of, B Lymphocyte Stimulator polypeptides with one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n¹-m¹ of SEQ ID NO:3228, where n¹ and m¹ are integers as defined above. Also included are antibodies that bind a polypeptide comprising, or alternatively consisting of, a portion of the complete B Lymphocyte Stimulator amino acid sequence encoded by the deposited cDNA clone contained in ATCC™ Accession No. 97768 where this portion excludes from 1 to 190 amino acids from the amino terminus or from 1 to 11 amino acids from the C-terminus of the complete amino acid sequence (or any combination of these N-terminal and C-terminal deletions) encoded by the cDNA clone in the deposited plasmid.

Similarly, deletions of C-terminal amino acid residues of the predicted extracellular domain of B Lymphocyte Stimulator up to the leucine residue at position 79 of SEQ ID NO:3228 may retain some biological activity, such as, for example, ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and modulation of cell replication or modulation of target cell activities. Polypeptides having further C-terminal deletions including Leu-79 of SEQ ID NO:3228 would not be expected to retain biological activities.

However, even if deletion of one or more amino acids from the C-terminus of a polypeptide results in modification or loss of one or more biological functions of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete, mature or extracellular forms of the polypeptide generally will be retained when less than the majority of the residues of the complete, mature or extracellular forms of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of the predicted extracellular domain retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the predicted extracellular domain of B Lymphocyte Stimulator polypeptide shown in SEQ ID NO:3228, up to the leucine residue at position 79 of SEQ ID NO:3228. In particular, the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 73-m² of the amino acid sequence in SEQ ID NO:3228, where m² is any integer in the range of the amino acid position of amino acid residues 79 to 285 in the amino acid sequence in SEQ ID NO:3228, and residue 78 is the position of the first residue at the C-terminus of the predicted extracellular domain of the B Lymphocyte Stimulator polypeptide (disclosed in SEQ ID NO:3228). More in particular, in certain embodiments, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues Q-73 to Leu-285; Q-73 to L-284; Q-73 to K-283; Q-73 to L-282; Q-73 to A-281; Q-73 to G-280; Q-73 to F-279; Q-73 to F-278; Q-73 to T-277; Q-73 to V-276; Q-73 to D-275; Q-73 to G-274; Q-73 to D-273; Q-73 to L-272; Q-73 to S-271; Q-73 to I-270; Q-73 to Q-269; Q-73 to A-268; Q-73 to N-267; Q-73 to E-266; Q-73 to R-265; Q-73 to P-264; Q-73 to I-263; Q-73 to A-262; Q-73 to L-261; Q-73 to Q-260; Q-73 to L-259; Q-73 to E-258; Q-73 to D-257; Q-73 to G-256; Q-73 to E-255; Q-73 to E-254; Q-73 to L-253; Q-73 to K-252; Q-73 to A-251; Q-73 to I-250; Q-73 to G-249; Q-73 to A-248; Q-73 to S-247; Q-73 to Y-246; Q-73 to C-245; Q-73 to S-244; Q-73 to N-243; Q-73 to N-242; Q-73 to P-241; Q-73 to L-240; Q-73 to T-239; Q-73 to E-238; Q-73 to P-237; Q-73 to M-236; Q-73 to N-235; Q-73 to Q-234; Q-73 to I-233; Q-73 to C-232; Q-73 to R-231; Q-73 to F-230; Q-73 to L-229; Q-73 to T-228; Q-73 to V-227; Q-73 to L-226; Q-73 to S-225; Q-73 to L-224; Q-73 to E-223; Q-73 to D-222; Q-73 to G-221; Q-73 to F-220; Q-73 to V-219; Q-73 to H-218; Q-73 to V-217; Q-73 to K-216; Q-73 to K-215; Q-73 to R-214; Q-73 to Q-213; Q-73 to I-212; Q-73 to L-211; Q-73 to H-210; Q-73 to G-209; Q-73 to M-208; Q-73 to A-207; Q-73 to Y-206; Q-73 to T-205; Q-73 to K-204; Q-73 to D-203; Q-73 to T-202; Q-73 to Y-201; Q-73 to L-200; Q-73 to V-199; Q-73 to Q-198; Q-73 to G-197; Q-73 to Y-196; Q-73 to I-195; Q-73 to F-194; Q-73 to F-193; Q-73 to Y-192; Q-73 to G-191; Q-73 to T-190; Q-73 to E-189; Q-73 to K-188; Q-73 to V-187; Q-73 to L-186; Q-73 to I-185; Q-73 to K-184; Q-73 to N-183; Q-73 to E-182; Q-73 to K-181; Q-73 to E-180; Q-73 to E-179; Q-73 to L-178; Q-73 to A-177; Q-73 to S-176; Q-73 to G-175; Q-73 to R-174; Q-73 to K-173; Q-73 to F-172; Q-73 to S-171; Q-73 to L-170; Q-73 to L-169; Q-73 to W-168; Q-73 to P-167; Q-73 to V-166; Q-73 to F-165; Q-73 to T-164; Q-73 to Y-163; Q-73 to S-162; Q-73 to G-161; Q-73 to K-160; Q-73 to Q-159; Q-73 to I-158; Q-73 to T-157; Q-73 to P-156; Q-73 to T-155; Q-73 to E-154; Q-73 to S-153; Q-73 to D-152; Q-73 to A-151; Q-73 to I-150; Q-73 to L-149; Q-73 to Q-148; Q-73 to L-147; Q-73 to C-146; Q-73 to D-145; Q-73 to Q-144; Q-73 to T-143; Q-73 to V-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; and Q-73 to L-79 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

The invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of the predicted extracellular domain of B Lymphocyte Stimulator, which may be described generally as having residues n²-m² of SEQ ID NO:3228 where n² and m² are integers as defined above.

In another embodiment, antibodies of the present invention bind polypeptides consisting of a portion of the extracellular domain of the B Lymphocyte Stimulator amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCC™ accession no. 97768, where this portion excludes from 1 to about 206 amino acids from the amino terminus of the extracellular domain of the amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCC™ accession no. 97768, or from 1 to about 206 amino acids from the carboxy terminus of the extracellular domain of the amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCC™ accession no. 97768, or any combination of the above amino terminal and carboxy terminal deletions, of the entire extracellular domain of the amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCC™ accession no. 97768.

As mentioned above, even if deletion of one or more amino acids from the N-terminus of a polypeptide results in modification or loss of one or more functional activities (e.g., biological activity) of the polypeptide, other functions or biological activities may still be retained. Thus, the ability of a shortened B Lymphocyte Stimulator mutein to induce and/or bind to antibodies which recognize the full-length or mature forms or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the full-length or mature or extracellular domain of the polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a B Lymphocyte Stimulator mutein with a large number of deleted N-terminal amino acid residues may retain some functional (e.g., biological or immunogenic) activities. In fact, peptides composed of as few as six B Lymphocyte Stimulator amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the predicted full-length amino acid sequence of the B Lymphocyte Stimulator shown in SEQ ID NO:3228, up to the glycine residue at position number 280 of the sequence shown SEQ ID NO:3228 and polynucleotides encoding such polypeptides. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n³-285 of the sequence shown in SEQ ID NO:3228, where n³ is an integer in the range of the amino acid position of amino acid residues 1 to 280 of the amino acid sequence in SEQ ID NO:3228.

More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of D-2 to L-285; D-3 to L-285; S-4 to L-285; T-5 to L-285; E-6 to L-285; R-7 to L-285; E-8 to L-285; Q-9 to L-285; S-10 to L-285; R-11 to L-285; L-12 to L-285; T-13 to L-285; S-14 to L-285; C-15 to L-285; L-16 to L-285; K-17 to L-285; K-18 to L-285; R-19 to L-285; E-20 to L-285; E-21 to L-285; M-22 to L-285; K-23 to L-285; L-24 to L-285; K-25 to L-285; E-26 to L-285; C-27 to L-285; V-28 to L-285; S-29 to L-285; I-30 to L-285; L-31 to L-285; P-32 to L-285; R-33 to L-285; K-34 to L-285; E-35 to L-285; S-36 to L-285; P-37 to L-285; S-38 to L-285; V-39 to L-285; R-40 to L-285; S-41 to L-285; S-42 to L-285; K-43 to L-285; D-44 to L-285; G-45 to L-285; K-46 to L-285; L-47 to L-285; L-48 to L-285; A-49 to L-285; A-50 to L-285; T-51 to L-285; L-52 to L-285; L-53 to L-285; L-54 to L-285; A-55 to L-285; L-56 to L-285; L-57 to L-285; S-58 to L-285; C-59 to L-285; C-60 to L-285; L-61 to L-285; T-62 to L-285; V-63 to L-285; V-64 to L-285; S-65 to L-285; F-66 to L-285; Y-67 to L-285; Q-68 to L-285; V-69 to L-285; A-70 to L-285; A-71 to L-285; L-72 to L-285; Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285; and G-280 to L-285 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more functional activities (e.g., biological activity) of the protein, other functional activities may still be retained. Thus, the ability of a shortened B Lymphocyte Stimulator mutein to induce and/or bind to antibodies which recognize the complete or mature form or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature form or the extracellular domain of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a B Lymphocyte Stimulator mutein with a large number of deleted C-terminal amino acid residues may retain some functional (e.g., biological or immunogenic) activities. In fact, peptides composed of as few as six B Lymphocyte Stimulator amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides in another embodiment, antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the B Lymphocyte Stimulator shown in SEQ ID NO:3228, up to the glutamic acid residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 1-m³ of SEQ ID NO:3228, where m³ is an integer in the range of the amino acid position of amino acid residues 6-284 of the amino acid sequence in SEQ ID NO:3228.

More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues M-1 to L-284; M-1 to K-283; M-1 to L-282; M-1 to A-281; M-1 to G-280; M-1 to F-279; M-1 to F-278; M-1 to T-277; M-1 to V-276; M-1 to D-275; M-1 to G-274; M-1 to D-273; M-1 to L-272; M-1 to S-271; M-1 to I-270; M-1 to Q-269; M-1 to A-268; M-1 to N-267; M-1 to E-266; M-1 to R-265; M-1 to P-264; M-1 to I-263; M-1 to A-262; M-1 to L-261; M-1 to Q-260; M-1 to L-259; M-1 to E-258; M-1 to D-257; M-1 to G-256; M-1 to E-255; M-1 to E-254; M-1 to L-253; M-1 to K-252; M-1 to A-251; M-1 to I-250; M-1 to G-249; M-1 to A-248; M-1 to S-247; M-1 to Y-246; M-1 to C-245; M-1 to S-244; M-1 to N-243; M-1 to N-242; M-1 to P-241; M-1 to L-240; M-1 to T-239; M-1 to E-238; M-1 to P-237; M-1 to M-236; M-1 to N-235; M-1 to Q-234; M-1 to I-233; M-1 to C-232; M-1 to R-231; M-1 to F-230; M-1 to L-229; M-1 to T-228; M-1 to V-227; M-1 to L-226; M-1 to S-225; M-1 to L-224; M-1 to E-223; M-1 to D-222; M-1 to G-221; M-1 to F-220; M-1 to V-219; M-1 to H-218; M-1 to V-217; M-1 to K-216; M-1 to K-215; M-1 to R-214; M-1 to Q-213; M-1 to I-212; M-1 to L-211; M-1 to H-210; M-1 to G-209; M-1 to M-208; M-1 to A-207; M-1 to Y-206; M-1 to T-205; M-1 to K-204; M-1 to D-203; M-1 to T-202; M-1 to Y-201; M-1 to L-200; M-1 to V-199; M-1 to Q-198; M-1 to G-197; M-1 to Y-196; M-1 to I-195; M-1 to F-194; M-1 to F-193; M-1 to Y-192; M-1 to G-191; M-1 to T-190; M-1 to E-189; M-1 to K-188; M-1 to V-187; M-1 to L-186; M-1 to I-185; M-1 to K-184; M-1 to N-183; M-1 to E-182; M-1 to K-181; M-1 to E-180; M-1 to E-179; M-1 to L-178; M-1 to A-177; M-1 to S-176; M-1 to G-175; M-1 to R-174; M-1 to K-173; M-1 to F-172; M-1 to S-171; M-1 to L-170; M-1 to L-169; M-1 to W-168; M-1 to P-167; M-1 to V-166; M-1 to F-165; M-1 to T-164; M-1 to Y-163; M-1 to S-162; M-1 to G-161; M-1 to K-160; M-1 to Q-159; M-1 to I-158; M-1 to T-157; M-1 to P-156; M-1 to T-155; M-1 to E-154; M-1 to S-153; M-1 to D-152; M-1 to A-151; M-1 to I-150; M-1 to L-149; M-1 to Q-148; M-1 to L-147; M-1 to C-146; M-1 to D-145; M-1 to Q-144; M-1 to T-143; M-1 to V-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M-1 to R-7; and M-1 to E-6 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

The invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a B Lymphocyte Stimulator polypeptide, which may be described generally as having residues n³-m³ of SEQ ID NO:3228, where n³ and m³ are integers as defined above.

Furthermore, since the predicted extracellular domain of the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3229 may itself elicit functional activity (e.g., biological activity), deletions of N- and C-terminal amino acid residues from the predicted extracellular region of the polypeptide at positions Gln-73 to Leu-266 of SEQ ID NO:3229 may retain some functional activity, such as, for example, ligand binding, to stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, modulation of cell replication, modulation of target cell activities and/or immunogenicity. However, even if deletion of one or more amino acids from the N-terminus of the predicted extracellular domain of a B Lymphocyte Stimulator polypeptide results in modification or loss of one or more functional activities of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptides to induce and/or bind to antibodies which recognize the complete or mature or extracellular domains of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature or extracellular domains of the polypeptides are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of B Lymphocyte Stimulator shown in SEQ ID NO:3229, up to the glycine residue at position number 261. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n⁴-266 of SEQ ID NO:3229, where n⁴ is an integer in the range of the amino acid position of amino acid residues 73-261 of the amino acid sequence in SEQ ID NO:3229, and 261 is the position of the first residue from the N-terminus of the predicted extracellular domain B Lymphocyte Stimulator polypeptide (shown in SEQ ID NO:3229).

More in particular, in certain embodiments, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of Q-73 to L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266; and G-261 to L-266 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

Similarly, deletions of C-terminal amino acid residues of the predicted extracellular domain of B Lymphocyte Stimulator up to the leucine residue at position 79 of SEQ ID NO:3229 may retain some functional activity, such as, for example, ligand binding, the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, modulation of cell replication, modulation of target cell activities and/or immunogenicity. Polypeptides having further C-terminal deletions including Leu-79 of SEQ ID NO:3229 would not be expected to retain biological activities.

However, even if deletion of one or more amino acids from the C-terminus of a polypeptide results in modification or loss of one or more functional activities (e.g., biological activity) of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete, mature or extracellular forms of the polypeptide generally will be retained when less than the majority of the residues of the complete, mature or extracellular forms of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of the predicted extracellular domain retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of the predicted extracellular domain of B Lymphocyte Stimulator shown in SEQ ID NO:3229, up to the leucine residue at position 79 of SEQ ID NO:3229. In particular, the present invention provides antibodies that bind polypeptides having the amino acid sequence of residues 73-m⁴ of the amino acid sequence in SEQ ID NO:3229, where m⁴ is any integer in the range of the amino acid position of amino acid residues 79-265 of the amino acid sequence in SEQ ID NO:3229.

More in particular, in certain embodiments, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues Q-73 to L-265; Q-73 to K-264; Q-73 to L-263; Q-73 to A-262; Q-73 to G-261; Q-73 to F-260; Q-73 to F-259; Q-73 to T-258; Q-73 to V-257; Q-73 to D-256; Q-73 to G-255; Q-73 to D-254; Q-73 to L-253; Q-73 to S-252; Q-73 to I-251; Q-73 to Q-250; Q-73 to A-249; Q-73 to N-248; Q-73 to E-247; Q-73 to R-246; Q-73 to P-245; Q-73 to I-244; Q-73 to A-243; Q-73 to L-242; Q-73 to Q-241; Q-73 to L-240; Q-73 to E-239; Q-73 to D-238; Q-73 to G-237; Q-73 to E-236; Q-73 to E-235; Q-73 to L-234; Q-73 to K-233; Q-73 to A-232; Q-73 to I-231; Q-73 to G-230; Q-73 to A-229; Q-73 to S-228; Q-73 to Y-227; Q-73 to C-226; Q-73 to S-225; Q-73 to N-224; Q-73 to N-223; Q-73 to P-222; Q-73 to L-221; Q-73 to T-220; Q-73 to E-219; Q-73 to P-218; Q-73 to M-217; Q-73 to N-216; Q-73 to Q-215; Q-73 to I-214; Q-73 to C-213; Q-73 to R-212; Q-73 to F-211; Q-73 to L-210; Q-73 to T-209; Q-73 to V-208; Q-73 to L-207; Q-73 to S-206; Q-73 to L-205; Q-73 to E-204; Q-73 to D-203; Q-73 to G-202; Q-73 to F-201; Q-73 to V-200; Q-73 to H-199; Q-73 to V-198; Q-73 to K-197; Q-73 to K-196; Q-73 to R-195; Q-73 to Q-194; Q-73 to I-193; Q-73 to L-192; Q-73 to H-191; Q-73 to G-190; Q-73 to Q-7389; Q-73 to A-188; Q-73 to Y-187; Q-73 to T-186; Q-73 to K-185; Q-73 to D-184; Q-73 to T-183; Q-73 to Y-182; Q-73 to L-181; Q-73 to V-180; Q-73 to Q-179; Q-73 to G-178; Q-73 to Y-177; Q-73 to I-176; Q-73 to F-175; Q-73 to F-174; Q-73 to Y-173; Q-73 to G-172; Q-73 to T-171; Q-73 to E-170; Q-73 to K-169; Q-73 to V-168; Q-73 to L-167; Q-73 to I-166; Q-73 to K-165; Q-73 to N-164; Q-73 to E-163; Q-73 to K-162; Q-73 to E-161; Q-73 to E-160; Q-73 to L-159; Q-73 to A-158; Q-73 to S-157; Q-73 to G-156; Q-73 to R-155; Q-73 to K-154; Q-73 to F-153; Q-73 to S-152; Q-73 to L-151; Q-73 to L-150; Q-73 to W-149; Q-73 to P-148; Q-73 to V-147; Q-73 to F-146; Q-73 to T-145; Q-73 to Y-144; Q-73 to S-143; Q-73 to G-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; Q-73 to L-79; and Q-73 to S-78 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of the predicted extracellular domain of B Lymphocyte Stimulator, which may be described generally as having residues n⁴-m⁴ of SEQ ID NO:3229 where n⁴ and m⁴ are integers as defined above.

In another embodiment, antibodies of the present invention bind polypeptides consisting of a portion of the extracellular domain of the B Lymphocyte Stimulator amino acid sequence encoded by the cDNA clone contained in the deposit having ATCC™ Accession No. 203518, where this portion excludes from 1 to about 260 amino acids from the amino terminus of the extracellular domain of the amino acid sequence encoded by cDNA clone contained in the deposit having ATCC™ Accession No. 203518, or from 1 to about 187 amino acids from the carboxy terminus of the extracellular domain of the amino acid sequence encoded by cDNA clone contained in the deposit having ATCC™ Accession No. 203518, or any combination of the above amino terminal and carboxy terminal deletions, of the entire extracellular domain of the amino acid sequence encoded by the cDNA clone contained in the deposit having ATCC™ Accession No. 203518.

As mentioned above, even if deletion of one or more amino acids from the N-terminus of a polypeptide results in modification or loss of one or more functional activities (e.g., biological activity) of the polypeptide, other functional activities may still be retained. Thus, the ability of a shortened B Lymphocyte Stimulator polypeptide to induce and/or bind to antibodies which recognize the full-length or mature forms or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the full-length or mature or extracellular domain of the polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a B Lymphocyte Stimulator mutein with a large number of deleted N-terminal amino acid residues may retain functional (e.g., immunogenic) activities. In fact, peptides composed of as few as six B Lymphocyte Stimulator amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the predicted full-length amino acid sequence of the B Lymphocyte Stimulator polypeptide shown in SEQ ID NO:3229, up to the glycine residue at position number 261 of the sequence shown SEQ ID NO:3229 and polynucleotides encoding such polypeptides. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n⁵-266 of the sequence shown in SEQ ID NO:3229, where n⁵ is an integer in the range of the amino acid position of amino acid residues 1 to 261 of the amino acid sequence in SEQ ID NO:3229.

More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of D-2 to L-266; D-3 to L-266; S-4 to L-266; T-5 to L-266; E-6 to L-266; R-7 to L-266; E-8 to L-266; Q-9 to L-266; S-10 to L-266; R-11 to L-266; L-12 to L-266; T-13 to L-266; S-14 to L-266; C-15 to L-266; L-16 to L-266; K-17 to L-266; K-18 to L-266; R-19 to L-266; E-20 to L-266; E-21 to L-266; M-22 to L-266; K-23 to L-266; L-24 to L-266; K-25 to L-266; E-26 to L-266; C-27 to L-266; V-28 to L-266; S-29 to L-266; I-30 to L-266; L-31 to L-266; P-32 to L-266; R-33 to L-266; K-34 to L-266; E-35 to L-266; S-36 to L-266; P-37 to L-266; S-38 to L-266; V-39 to L-266; R-40 to L-266; S-41 to L-266; S-42 to L-266; K-43 to L-266; D-44 to L-266; G-45 to L-266; K-46 to L-266; L-47 to L-266; L-48 to L-266; A-49 to L-266; A-50 to L-266; T-51 to L-266; L-52 to L-266; L-53 to L-266; L-54 to L-266; A-55 to L-266; L-56 to L-266; L-57 to L-266; S-58 to L-266; C-59 to L-266; C-60 to L-266; L-61 to L-266; T-62 to L-266; V-63 to L-266; V-64 to L-266; S-65 to L-266; F-66 to L-266; Y-67 to L-266; Q-68 to L-266; V-69 to L-266; A-70 to L-266; A-71 to L-266; L-72 to L-266; Q-73 to L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266; and G-261 to L-266 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more functional activities (e.g., biological activities) of the protein, other functional activities may still be retained. Thus, the ability of a shortened B Lymphocyte Stimulator mutein to induce and/or bind to antibodies which recognize the complete or mature form or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature form or the extracellular domain of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a B Lymphocyte Stimulator mutein with a large number of deleted C-terminal amino acid residues may retain some functional (e.g., immunogenic) activities. In fact, peptides composed of as few as six B Lymphocyte Stimulator amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides in another embodiment, antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the B Lymphocyte Stimulator shown in SEQ ID NO:3229, up to the glutamic acid residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 1-m⁵ of SEQ ID NO:3229, where m⁵ is an integer in the range of the amino acid position of amino acid residues 6 to 265 in the amino acid sequence of SEQ ID NO:3229.

More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues M-1 to L-265; M-1 to K-264; M-1 to L-263; M-1 to A-262; M-1 to G-261; M-1 to F-260; M-1 to F-259; M-1 to T-258; M-1 to V-257; M-1 to D-256; M-1 to G-255; M-1 to D-254; M-1 to L-253; M-1 to S-252; M-1 to I-251; M-1 to Q-250; M-1 to A-249; M-1 to N-248; M-1 to E-247; M-1 to R-246; M-1 to P-245; M-1 to I-244; M-1 to A-243; M-1 to L-242; M-1 to Q-241; M-1 to L-240; M-1 to E-239; M-1 to D-238; M-1 to G-237; M-1 to E-236; M-1 to E-235; M-1 to L-234; M-1 to K-233; M-1 to A-232; M-1 to I-231; M-1 to G-230; M-1 to A-229; M-1 to S-228; M-1 to Y-227; M-1 to C-226; M-1 to S-225; M-1 to N-224; M-1 to N-223; M-1 to P-222; M-1 to L-221; M-1 to T-220; M-1 to E-219; M-1 to P-218; M-1 to M-217; M-1 to N-216; M-1 to Q-215; M-1 to I-214; M-1 to C-213; M-1 to R-212; M-1 to F-211; M-1 to L-210; M-1 to T-209; M-1 to V-208; M-1 to L-207; M-1 to S-206; M-1 to L-205; M-1 to E-204; M-1 to D-203; M-1 to G-202; M-1 to F-201; M-1 to V-200; M-1 to H-199; M-1 to V-198; M-1 to K-197; M-1 to K-196; M-1 to R-195; M-1 to Q-194; M-1 to I-193; M-1 to L-192; M-1 to H-191; M-1 to G-190; M-1 to M-189; M-1 to A-188; M-1 to Y-187; M-1 to T-186; M-1 to K-185; M-1 to D-184; M-1 to T-183; M-1 to Y-182; M-1 to L-181; M-1 to V-180; M-1 to Q-179; M-1 to G-178; M-1 to Y-177; M-1 to I-176; M-1 to F-175; M-1 to F-174; M-1 to Y-173; M-1 to G-172; M-1 to T-171; M-1 to E-170; M-1 to K-169; M-1 to V-168; M-1 to L-167; M-1 to I-166; M-1 to K-165; M-1 to N-164; M-1 to E-163; M-1 to K-162; M-1 to E-161; M-1 to E-160; M-1 to L-159; M-1 to A-158; M-1 to S-157; M-1 to G-156; M-1 to R-155; M-1 to K-154; M-1 to F-153; M-1 to S-152; M-1 to L-151; M-1 to L-150; M-1 to W-149; M-1 to P-148; M-1 to V-147; M-1 to F-146; M-1 to T-145; M-1 to Y-144; M-1 to S-143; M-1 to G-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M-1 to R-7; and M-1 to E-6 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

The invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a B Lymphocyte Stimulator polypeptide, which may be described generally as having residues n⁵-m⁵ of SEQ ID NO:3229, where n⁵ and m⁵ are integers as defined above.

In additional embodiments, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 134-m⁶ of SEQ ID NO:3228, where m⁶ is an integer from 140 to 285, corresponding to the position of the amino acid residue in SEQ ID NO:3228. For example, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues A-134 to Leu-285; A-134 to L-284; A-134 to K-283; A-134 to L-282; A-134 to A-281; A-134 to G-280; A-134 to F-279; A-134 to F-278; A-134 to T-277; A-134 to V-276; A-134 to D-275; A-134 to G-274; A-134 to D-273; A-134 to L-272; A-134 to S-271; A-134 to I-270; A-134 to Q-269; A-134 to A-268; A-134 to N-267; A-134 to E-266; A-134 to R-265; A-134 to P-264; A-134 to I-263; A-134 to A-262; A-134 to L-261; A-134 to Q-260; A-134 to L-259; A-134 to E-258; A-134 to D-257; A-134 to G-256; A-134 to E-255; A-134 to E-254; A-134 to L-253; A-134 to K-252; A-134 to A-251; A-134 to I-250; A-134 to G-249; A-134 to A-248; A-134 to S-247; A-134 to Y-246; A-134 to C-245; A-134 to S-244; A-134 to N-243; A-134 to N-242; A-134 to P-241; A-134 to L-240; A-134 to T-239; A-134 to E-238; A-134 to P-237; A-134 to M-236; A-134 to N-235; A-134 to Q-234; A-134 to I-233; A-134 to C-232; A-134 to R-231; A-134 to F-230; A-134 to L-229; A-134 to T-228; A-134 to V-227; A-134 to L-226; A-134 to S-225; A-134 to L-224; A-134 to E-223; A-134 to D-222; A-134 to G-221; A-134 to F-220; A-134 to V-219; A-134 to H-218; A-134 to V-217; A-134 to K-216; A-134 to K-215; A-134 to R-214; A-134 to Q-213; A-134 to I-212; A-134 to L-211; A-134 to H-210; A-134 to G-209; A-134 to M-208; A-134 to A-207; A-134 to Y-206; A-134 to T-205; A-134 to K-204; A-134 to D-203; A-134 to T-202; A-134 to Y-201; A-134 to L-200; A-134 to V-199; A-134 to Q-198; A-134 to G-197; A-134 to Y-196; A-134 to I-195; A-134 to F-194; A-134 to F-193; A-134 to Y-192; A-134 to G-191; A-134 to T-190; A-134 to E-189; A-134 to K-188; A-134 to V-187; A-134 to L-186; A-134 to I-185; A-134 to K-184; A-134 to N-183; A-134 to E-182; A-134 to K-181; A-134 to E-180; A-134 to E-179; A-134 to L-178; A-134 to A-177; A-134 to S-176; A-134 to G-175; A-134 to R-174; A-134 to K-173; A-134 to F-172; A-134 to S-171; A-134 to L-170; A-134 to L-169; A-134 to W-168; A-134 to P-167; A-134 to V-166; A-134 to F-165; A-134 to T-164; A-134 to Y-163; A-134 to S-162; A-134 to G-161; A-134 to K-160; A-134 to Q-159; A-134 to I-158; A-134 to T-157; A-134 to P-156; A-134 to T-155; A-134 to E-154; A-134 to S-153; A-134 to D-152; A-134 to A-151; A-134 to I-150; A-134 to L-149; A-134 to Q-148; A-134 to L-147; A-134 to C-146; A-134 to D-145; A-134 to Q-144; A-134 to T-143; A-134 to V-142; A-134 to T-141; and A-134 to E-140 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

In additional embodiments, antibodies of the present invention may bind polypeptide fragments comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues: M-1 to C-15; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; S-42 to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; A-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to V-142; S-129 to T-143; R-130 to Q-144; N-131 to D-145; K-132 to C-146; R-133 to L-147; A-134 to Q-148; V-135 to L-149; Q-136 to I-150; G-137 to A-151; P-138 to D-152; E-139 to S-153; E-140 to E-154; T-141 to T-155; V-142 to P-156; T-143 to T-157; Q-144 to I-158; D-145 to Q-159; C-146 to K-160; L-147 to G-161; Q-148 to S-162; L-149 to Y-163; I-150 to T-164; A-151 to F-165; D-152 to V-166; S-153 to P-167; E-154 to W-168; T-155 to L-169; P-156 to L-170; T-157 to S-171; I-158 to F-172; Q-159 to K-173; K-160 to R-174; G-161 to G-175; S-162 to S-176; Y-163 to A-177; T-164 to L-178; F-165 to E-179; V-166 to E-180; P-167 to K-181; W-168 to E-182; L-169 to N-183; L-170 to K-184; S-171 to I-185; F-172 to L-186; K-173 to V-187; R-174 to K-188; G-175 to E-189; S-176 to T-190; A-177 to G-191; L-178 to Y-192; E-179 to F-193; E-180 to F-194; K-181 to I-195; E-182 to Y-196; N-183 to G-197; K-184 to Q-198; I-185 to V-199; L-186 to L-200; V-187 to Y-201; K-188 to T-202; E-189 to D-203; T-190 to K-204; G-191 to T-205; Y-192 to Y-206; F-193 to A-207; F-194 to M-208; I-195 to G-209; Y-196 to H-210; G-197 to L-211; Q-198 to I-212; V-199 to Q-213; L-200 to R-214; Y-201 to K-215; T-202 to K-216; D-203 to V-217; K-204 to H-218; T-205 to V-219; Y-206 to F-220; A-207 to G-221; M-208 to D-222; G-209 to E-223; H-210 to L-224; L-211 to S-225; I-212 to L-226; Q-213 to V-227; R-214 to T-228; K-215 to L-229; K-216 to F-230; V-217 to R-231; H-218 to C-232; V-219 to I-233; F-220 to Q-234; G-221 to N-235; D-222 to M-236; E-223 to P-237; L-224 to E-238; S-225 to T-239; L-226 to L-240; V-227 to P-241; T-228 to N-242; L-229 to N-243; F-230 to S-244; R-231 to C-245; C-232 to Y-246; I-233 to S-247; Q-234 to A-248; N-235 to G-249; M-236 to I-250; P-237 to A-251; E-238 to K-252; T-239 to L-253; L-240 to E-254; P-241 to E-255; N-242 to G-256; N-243 to D-257; S-244 to E-258; C-245 to L-259; Y-246 to Q-260; S-247 to L-261; A-248 to A-262; G-249 to I-263; I-250 to P-264; A-251 to R-265; K-252 to E-266; L-253 to N-267; E-254 to A-268; E-255 to Q-269; G-256 to I-270; D-257 to S-271; E-258 to L-272; L-259 to D-273; Q-260 to G-274; L-261 to D-275; A-262 to V-276; I-263 to T-277; P-264 to F-278; R-265 to F-279; E-266 to G-280; N-267 to A-281; A-268 to L-282; Q-269 to K-283; I-270 to L-284; and S-271 to L-285 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

In additional embodiments, antibodies of the present invention may bind polypeptide fragments comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues: M-1 to C-15; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; S-42 to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; A-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to G-142; S-129 to S-143; R-130 to Y-144; N-131 to T-145; K-132 to F-146; R-133 to V-147; A-134 to P-148; V-135 to W-149; Q-136 to L-150; G-137 to L-151; P-138 to S-152; E-139 to F-153; E-140 to K-154; T-141 to R-155; G-142 to G-156; S-143 to S-157; Y-144 to A-158; T-145 to L-159; F-146 to E-160; V-147 to E-161; P-148 to K-162; W-149 to E-163; L-150 to N-164; L-151 to K-165; S-152 to I-166; F-153 to L-167; K-154 to V-168; R-155 to K-169; G-156 to E-170; S-157 to T-171; A-158 to G-172; L-159 to Y-173; E-160 to F-174; E-161 to F-175; K-162 to I-176; E-163 to Y-177; N-164 to G-178; K-165 to Q-179; I-166 to V-180; L-167 to L-181; V-168 to Y-182; K-169 to T-183; E-170 to D-184; T-171 to K-185; G-172 to T-186; Y-173 to Y-187; F-174 to A-188; F-175 to M-189; I-176 to G-190; Y-177 to H-191; G-178 to L-192; Q-179 to I-193; V-180 to Q-194; L-181 to R-195; Y-182 to K-196; T-183 to K-197; D-184 to V-198; K-185 to H-199; T-186 to V-200; Y-187 to F-201; A-188 to G-202; M-189 to D-203; G-190 to E-204; H-191 to L-205; L-192 to S-206; I-193 to L-207; Q-194 to V-208; R-195 to T-209; K-196 to L-210; K-197 to F-211; V-198 to R-212; H-199 to C-213; V-200 to I-214; F-201 to Q-215; G-202 to N-216; D-203 to M-217; E-204 to P-218; L-205 to E-219; S-206 to T-220; L-207 to L-221; V-208 to P-222; T-209 to N-223; L-210 to N-224; F-211 to S-225; R-212 to C-226; C-213 to Y-227; I-214 to S-228; Q-215 to A-229; N-216 to G-230; M-217 to I-231; P-218 to A-232; E-219 to K-233; T-220 to L-234; L-221 to E-235; P-222 to E-236; N-223 to G-237; N-224 to D-238; S-225 to E-239; C-226 to L-240; Y-227 to Q-241; S-228 to L-242; A-229 to A-243; G-230 to I-244; I-231 to P-245; A-232 to R-246; K-233 to E-247; L-234 to N-248; E-235 to A-249; E-236 to Q-250; G-237 to I-251; D-238 to S-252; E-239 to L-253; L-240 to D-254; Q-241 to G-255; L-242 to D-256; A-243 to V-257; I-244 to T-258; P-245 to F-259; R-246 to F-260; E-247 to G-261; N-248 to A-262; A-249 to L-263; Q-250 to K-264; I-251 to L-265; and S-252 to L-266 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

In additional embodiments, antibodies of the present invention may bind polypeptide fragments comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues: M-1 to F-15; D-2 to C-16; E-3 to S-17; S-4 to E-18; A-5 to K-19; K-6 to G-20; T-7 to E-21; L-8 to D-22; P-9 to M-23; P-10 to K-24; P-11 to V-25; C-12 to G-26; L-13 to Y-27; C-14 to D-28; F-15 to P-29; C-16 to I-30; S-17 to T-31; E-18 to P-32; K-19 to Q-33; G-20 to K-34; E-21 to E-35; D-22 to E-36; M-23 to G-37; K-24 to A-38; V-25 to W-39; G-26 to F-40; Y-27 to G-41; D-28 to I-42; P-29 to C-43; I-30 to R-44; T-31 to D-45; P-32 to G-46; Q-33 to R-47; K-34 to L-48; E-35 to L-49; E-36 to A-50; G-37 to A-51; A-38 to T-52; W-39 to L-53; F-40 to L-54; G-41 to L-55; I-42 to A-56; C-43 to L-57; R-44 to L-58; D-45 to S-59; G-46 to S-60; R-47 to S-61; L-48 to F-62; L-49 to T-63; A-50 to A-64; A-51 to M-65; T-52 to S-66; L-53 to L-67; L-54 to Y-68; L-55 to Q-69; A-56 to L-70; L-57 to A-71; L-58 to A-72; S-59 to L-73; S-60 to Q-74; S-61 to A-75; F-62 to D-76; T-63 to L-77; A-64 to M-78; M-65 to N-79; S-66 to L-80; L-67 to R-81; Y-68 to M-82; Q-69 to E-83; L-70 to L-84; A-71 to Q-85; A-72 to S-86; L-73 to Y-87; Q-74 to R-88; A-75 to G-89; D-76 to S-90; L-77 to A-91; M-78 to T-92; N-79 to P-93; L-80 to A-94; R-81 to A-95; M-82 to A-96; E-83 to G-97; L-84 to A-98; Q-85 to P-99; S-86 to E-100; Y-87 to L-101; R-88 to T-102; G-89 to A-103; S-90 to G-104; A-91 to V-105; T-92 to K-106; P-93 to L-107; A-94 to L-108; A-95 to T-109; A-96 to P-110; G-97 to A-111; A-98 to A-112; P-99 to P-113; E-100 to R-114; L-101 to P-115; T-102 to H-116; A-103 to N-117; G-104 to S-118; V-105 to S-119; K-106 to R-120; L-107 to G-121; L-108 to H-122; T-109 to R-123; P-110 to N-124; A-111 to R-125; A-112 to R-126; P-113 to A-127; R-114 to F-128; P-115 to Q-129; H-116 to G-130; N-117 to P-131; S-118 to E-132; S-119 to E-133; R-120 to T-134; G-121 to E-135; H-122 to Q-136; R-123 to D-137; N-124 to V-138; R-125 to D-139; R-126 to L-140; A-127 to S-141; F-128 to A-142; Q-129 to P-143; G-130 to P-144; P-131 to A-145; E-132 to P-146; E-133 to C-147; T-134 to L-148; E-135 to P-149; Q-136 to G-150; D-137 to C-151; V-138 to R-152; D-139 to H-153; L-140 to S-154; S-141 to Q-155; A-142 to H-156; P-143 to D-157; P-144 to D-158; A-145 to N-159; P-146 to G-160; C-147 to M-161; L-148 to N-162; P-149 to L-163; G-150 to R-164; C-151 to N-165; R-152 to I-166; H-153 to I-167; S-154 to Q-168; Q-155 to D-169; H-156 to C-170; D-157 to L-171; D-158 to Q-172; N-159 to L-173; G-160 to I-174; M-161 to A-175; N-162 to D-176; L-163 to S-177; R-164 to D-178; N-165 to T-179; I-166 to P-180; I-167 to A-181; Q-168 to L-182; D-169 to E-183; C-170 to E-184; L-171 to K-185; Q-172 to E-186; L-173 to N-187; I-174 to K-188; A-175 to I-189; D-176 to V-190; S-177 to V-191; D-178 to R-192; T-179 to Q-193; P-180 to T-194; A-181 to G-195; L-182 to Y-196; E-183 to F-197; E-184 to F-198; K-185 to I-199; E-186 to Y-200; N-187 to S-201; K-188 to Q-202; I-189 to V-203; V-190 to L-204; V-191 to Y-205; R-192 to T-206; Q-193 to D-207; T-194 to P-208; G-195 to I-209; Y-196 to F-210; F-197 to A-211; F-198 to M-212; I-199 to G-213; Y-200 to H-214; S-201 to V-215; Q-202 to I-216; V-203 to Q-217; L-204 to R-218; Y-205 to K-219; T-206 to K-220; D-207 to V-221; P-208 to H-222; I-209 to V-223; F-210 to F-224; A-211 to G-225; M-212 to D-226; G-213 to E-227; H-214 to L-228; V-215 to S-229; I-216 to L-230; Q-217 to V-231; R-218 to T-232; K-219 to L-233; K-220 to F-234; V-221 to R-235; H-222 to C-236; V-223 to I-237; F-224 to Q-238; G-225 to N-239; D-226 to M-240; E-227 to P-241; L-228 to K-242; S-229 to T-243; L-230 to L-244; V-231 to P-245; T-232 to N-246; L-233 to N-247; F-234 to S-248; R-235 to C-249; C-236 to Y-250; I-237 to S-251; Q-238 to A-252; N-239 to G-253; M-240 to I-254; P-241 to A-255; K-242 to R-256; T-243 to L-257; L-244 to E-258; P-245 to E-259; N-246 to G-260; N-247 to D-261; S-248 to E-262; C-249 to I-263; Y-250 to Q-264; S-251 to L-265; A-252 to A-266; G-253 to I-267; I-254 to P-268; A-255 to R-269; R-256 to E-270; L-257 to N-271; E-258 to A-272; E-259 to Q-273; G-260 to I-274; D-261 to S-275; E-262 to R-276; I-263 to N-277; Q-264 to G-278; L-265 to D-279; A-266 to D-280; I-267 to T-281; P-268 to F-282; R-269 to F-283; E-270 to G-284; N-271 to A-285; A-272 to L-286; Q-273 to K-287; I-274 to L-288; and S-275 to L-289 of SEQ ID NO:38. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.

It will be recognized by one of ordinary skill in the art that some amino acid sequences of the B Lymphocyte Stimulator polypeptides can be varied without significant effect of the structure or function of the polypeptide. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the polypeptide which determine activity.

Thus, the invention further includes antibodies that bind variations of B Lymphocyte Stimulator polypeptides which show B Lymphocyte Stimulator polypeptide functional activity (e.g., biological activity) or which include regions of B Lymphocyte Stimulator polypeptide such as the polypeptide fragments described herein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., “Deciphering the Message in Protein Sequences Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality.

As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie, J. U. et al., supra, and the references cited therein. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

Thus, antibodies of the present invention may bind fragments, derivatives or analogs of the polypeptide of SEQ ID NO:3228, or that encoded by the deposited cDNA plasmid, such as (i) polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) polypeptides in which one or more of the amino acid residues includes a substituent group, or (iii) polypeptides in which the extracellular domain of the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) polypeptides in which the additional amino acids are fused to the extracellular domain of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the extracellular domain of the polypeptide or a proprotein sequence.

Antibodies of the present invention may bind fragments, derivatives or analogs of the polypeptide of SEQ ID NO:3229, or that encoded by the deposited cDNA plasmid, such as (i) polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) polypeptides in which one or more of the amino acid residues includes a substituent group, or (iii) polypeptides in which the extracellular domain of the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) polypeptides in which the additional amino acids are fused to the extracellular domain of the polypeptide, such as, a soluble biologically active fragment of another TNF ligand family member (e.g., CD40 Ligand), an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the extracellular domain of the polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

Thus, the antibodies of the invention may bind B Lymphocyte Stimulator polypeptides that include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 13).

TABLE 13 Conservative Amino Acid Substitutions. Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

In one embodiment of the invention, antibodies of the present invention bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of a B Lymphocyte Stimulator polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions. In one embodiment of the invention, antibodies of the present invention bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of a B Lymphocyte Stimulator polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.

For example, site directed changes at the amino acid level of B Lymphocyte Stimulator can be made by replacing a particular amino acid with a conservative substitution. Antibodies of the present invention may bind B Lymphocyte Stimulator amino acid sequences containing conservative substitution mutations of the polypeptide of SEQ ID NO:3228 including: M1 replaced with A, G, I, L, S, T, or V; D2 replaced with E; D3 replaced with E; S4 replaced with A, G, I, L, T, M, or V; T5 replaced with A, G, I, L, S, M, or V; E6 replaced with D; R7 replaced with H, or K; E8 replaced with D; Q9 replaced with N; S10 replaced with A, G, I, L, T, M, or V; R11 replaced with H, or K; L12 replaced with A, G, I, S, T, M, or V; T13 replaced with A, G, I, L, S, M, or V; S14 replaced with A, G, I, L, T, M, or V; L16 replaced with A, G, I, S, T, M, or V; K17 replaced with H, or R; K18 replaced with H, or R; R19 replaced with H, or K; E20 replaced with D; E21 replaced with D; M22 replaced with A, G, I, L, S, T, or V; K23 replaced with H, or R; L24 replaced with A, G, I, S, T, M, or V; K25 replaced with H, or R; E26 replaced with D; V28 replaced with A, G, I, L, S, T, or M; S29 replaced with A, G, I, L, T, M, or V; I30 replaced with A, G, L, S, T, M, or V; L31 replaced with A, G, I, S, T, M, or V; R33 replaced with H, or K; K34 replaced with H, or R; E35 replaced with D; S36 replaced with A, G, I, L, T, M, or V; S38 replaced with A, G, I, L, T, M, or V; V39 replaced with A, G, I, L, S, T, or M; R40 replaced with H, or K; S41 replaced with A, G, I, L, T, M, or V; S42 replaced with A, G, I, L, T, M, or V; K43 replaced with H, or R; D44 replaced with E; G45 replaced with A, I, L, S, T, M, or V; K46 replaced with H, or R; L47 replaced with A, G, I, S, T, M, or V; L48 replaced with A, G, I, S, T, M, or V; A49 replaced with G, I, L, S, T, M, or V; A50 replaced with G, I, L, S, T, M, or V; T51 replaced with A, G, I, L, S, M, or V; L52 replaced with A, G, I, S, T, M, or V; L53 replaced with A, G, I, S, T, M, or V; L54 replaced with A, G, I, S, T, M, or V; A55 replaced with G, I, L, S, T, M, or V; L56 replaced with A, G, I, S, T, M, or V; L57 replaced with A, G, I, S, T, M, or V; S58 replaced with A, G, I, L, T, M, or V; L61 replaced with A, G, I, S, T, M, or V; T62 replaced with A, G, I, L, S, M, or V; V63 replaced with A, G, I, L, S, T, or M; V64 replaced with A, G, I, L, S, T, or M; S65 replaced with A, G, I, L, T, M, or V; F66 replaced with W, or Y; Y67 replaced with F, or W; Q68 replaced with N; V69 replaced with A, G, I, L, S, T, or M; A70 replaced with G, I, L, S, T, M, or V; A71 replaced with G, I, L, S, T, M, or V; L72 replaced with A, G, I, S, T, M, or V; Q73 replaced with N; G74 replaced with A, I, L, S, T, M, or V; D75 replaced with E; L76 replaced with A, G, I, S, T, M, or V; A77 replaced with G, I, L, S, T, M, or V; S78 replaced with A, G, I, L, T, M, or V; L79 replaced with A, G, I, S, T, M, or V; R80 replaced with H, or K; A81 replaced with G, I, L, S, T, M, or V; E82 replaced with D; L83 replaced with A, G, I, S, T, M, or V; Q84 replaced with N; G85 replaced with A, I, L, S, T, M, or V; H86 replaced with K, or R; H87 replaced with K, or R; A88 replaced with G, I, L, S, T, M, or V; E89 replaced with D; K90 replaced with H, or R; L91 replaced with A, G, I, S, T, M, or V; A93 replaced with G, I, L, S, T, M, or V; G94 replaced with A, I, L, S, T, M, or V; A95 replaced with G, I, L, S, T, M, or V; G96 replaced with A, I, L, S, T, M, or V; A97 replaced with G, I, L, S, T, M, or V; K99 replaced with H, or R; A100 replaced with G, I, L, S, T, M, or V; G101 replaced with A, I, L, S, T, M, or V; L102 replaced with A, G, I, S, T, M, or V; E103 replaced with D; E104 replaced with D; A105 replaced with G, I, L, S, T, M, or V; A107 replaced with G, I, L, S, T, M, or V; V108 replaced with A, G, I, L, S, T, or M; T109 replaced with A, G, I, L, S, M, or V; A110 replaced with G, I, L, S, T, M, or V; G111 replaced with A, I, L, S, T, M, or V; L112 replaced with A, G, I, S, T, M, or V; K113 replaced with H, or R; I114 replaced with A, G, L, S, T, M, or V; F115 replaced with W, or Y; E116 replaced with D; A119 replaced with G, I, L, S, T, M, or V; G121 replaced with A, I, L, S, T, M, or V; E122 replaced with D; G123 replaced with A, I, L, S, T, M, or V; N124 replaced with Q; S125 replaced with A, G, I, L, T, M, or V; S126 replaced with A, G, I, L, T, M, or V; Q127 replaced with N; N128 replaced with Q; S129 replaced with A, G, I, L, T, M, or V; R130 replaced with H, or K; N131 replaced with Q; K132 replaced with H, or R; R133 replaced with H, or K; A134 replaced with G, I, L, S, T, M, or V; V135 replaced with A, G, I, L, S, T, or M; Q136 replaced with N; G137 replaced with A, I, L, S, T, M, or V; E139 replaced with D; E140 replaced with D; T141 replaced with A, G, I, L, S, M, or V; V142 replaced with A, G, I, L, S, T, or M; T143 replaced with A, G, I, L, S, M, or V; Q144 replaced with N; D145 replaced with E; L147 replaced with A, G, I, S, T, M, or V; Q148 replaced with N; L149 replaced with A, G, I, S, T, M, or V; I150 replaced with A, G, L, S, T, M, or V; A151 replaced with G, I, L, S, T, M, or V; D152 replaced with E; S153 replaced with A, G, I, L, T, M, or V; E154 replaced with D; T155 replaced with A, G, I, L, S, M, or V; T157 replaced with A, G, I, L, S, M, or V; I158 replaced with A, G, L, S, T, M, or V; Q159 replaced with N; K160 replaced with H, or R; G161 replaced with A, I, L, S, T, M, or V; S162 replaced with A, G, I, L, T, M, or V; Y163 replaced with F, or W; T164 replaced with A, G, I, L, S, M, or V; F165 replaced with W, or Y; V166 replaced with A, G, I, L, S, T, or M; W168 replaced with F, or Y; L169 replaced with A, G, I, S, T, M, or V; L170 replaced with A, G, I, S, T, M, or V; S171 replaced with A, G, I, L, T, M, or V; F172 replaced with W, or Y; K173 replaced with H, or R; R174 replaced with H, or K; G175 replaced with A, I, L, S, T, M, or V; S176 replaced with A, G, I, L, T, M, or V; A177 replaced with G, I, L, S, T, M, or V; L178 replaced with A, G, I, S, T, M, or V; E179 replaced with D; E180 replaced with D; K181 replaced with H, or R; E182 replaced with D; N183 replaced with Q; K184 replaced with H, or R; I185 replaced with A, G, L, S, T, M, or V; L186 replaced with A, G, I, S, T, M, or V; V187 replaced with A, G, I, L, S, T, or M; K188 replaced with H, or R; E189 replaced with D; T190 replaced with A, G, I, L, S, M, or V; G191 replaced with A, I, L, S, T, M, or V; Y192 replaced with F, or W; F193 replaced with W, or Y; F194 replaced with W, or Y; I195 replaced with A, G, L, S, T, M, or V; Y196 replaced with F, or W; G197 replaced with A, I, L, S, T, M, or V; Q198 replaced with N; V199 replaced with A, G, I, L, S, T, or M; L200 replaced with A, G, I, S, T, M, or V; Y201 replaced with F, or W; T202 replaced with A, G, I, L, S, M, or V; D203 replaced with E; K204 replaced with H, or R; T205 replaced with A, G, I, L, S, M, or V; Y206 replaced with F, or W; A207 replaced with G, I, L, S, T, M, or V; M208 replaced with A, G, I, L, S, T, or V; G209 replaced with A, I, L, S, T, M, or V; H210 replaced with K, or R; L211 replaced with A, G, I, S, T, M, or V; I212 replaced with A, G, L, S, T, M, or V; Q213 replaced with N; R214 replaced with H, or K; K215 replaced with H, or R; K216 replaced with H, or R; V217 replaced with A, G, I, L, S, T, or M; H218 replaced with K, or R; V219 replaced with A, G, I, L, S, T, or M; F220 replaced with W, or Y; G221 replaced with A, I, L, S, T, M, or V; D222 replaced with E; E223 replaced with D; L224 replaced with A, G, I, S, T, M, or V; S225 replaced with A, G, I, L, T, M, or V; L226 replaced with A, G, I, S, T, M, or V; V227 replaced with A, G, I, L, S, T, or M; T228 replaced with A, G, I, L, S, M, or V; L229 replaced with A, G, I, S, T, M, or V; F230 replaced with W, or Y; R231 replaced with H, or K; I233 replaced with A, G, L, S, T, M, or V; Q234 replaced with N; N235 replaced with Q; M236 replaced with A, G, I, L, S, T, or V; E238 replaced with D; T239 replaced with A, G, I, L, S, M, or V; L240 replaced with A, G, I, S, T, M, or V; N242 replaced with Q; N243 replaced with Q; S244 replaced with A, G, I, L, T, M, or V; Y246 replaced with F, or W; S247 replaced with A, G, I, L, T, M, or V; A248 replaced with G, I, L, S, T, M, or V; G249 replaced with A, I, L, S, T, M, or V; I250 replaced with A, G, L, S, T, M, or V; A251 replaced with G, I, L, S, T, M, or V; K252 replaced with H, or R; L253 replaced with A, G, I, S, T, M, or V; E254 replaced with D; E255 replaced with D; G256 replaced with A, I, L, S, T, M, or V; D257 replaced with E; E258 replaced with D; L259 replaced with A, G, I, S, T, M, or V; Q260 replaced with N; L261 replaced with A, G, I, S, T, M, or V; A262 replaced with G, I, L, S, T, M, or V; I263 replaced with A, G, L, S, T, M, or V; R265 replaced with H, or K; E266 replaced with D; N267 replaced with Q; A268 replaced with G, I, L, S, T, M, or V; Q269 replaced with N; I270 replaced with A, G, L, S, T, M, or V; S271 replaced with A, G, I, L, T, M, or V; L272 replaced with A, G, I, S, T, M, or V; D273 replaced with E; G274 replaced with A, I, L, S, T, M, or V; D275 replaced with E; V276 replaced with A, G, I, L, S, T, or M; T277 replaced with A, G, I, L, S, M, or V; F278 replaced with W, or Y; F279 replaced with W, or Y; G280 replaced with A, I, L, S, T, M, or V; A281 replaced with G, I, L, S, T, M, or V; L282 replaced with A, G, I, S, T, M, or V; K283 replaced with H, or R; L284 replaced with A, G, I, S, T, M, or V; and/or L285 replaced with A, G, I, S, T, M, or V.

In another embodiment, site directed changes at the amino acid level of B Lymphocyte Stimulator can be made by replacing a particular amino acid with a conservative substitution. Antibodies of the present invention may bind B Lymphocyte Stimulator amino acid sequences containing conservative substitution mutations of the polypeptide of SEQ ID NO:3229 including: M1 replaced with A, G, I, L, S, T, or V; D2 replaced with E; D3 replaced with E; S4 replaced with A, G, I, L, T, M, or V; T5 replaced with A, G, I, L, S, M, or V; E6 replaced with D; R7 replaced with H, or K; E8 replaced with D; Q9 replaced with N; S10 replaced with A, G, I, L, T, M, or V; R11 replaced with H, or K; L12 replaced with A, G, I, S, T, M, or V; T13 replaced with A, G, I, L, S, M, or V; S14 replaced with A, G, I, L, T, M, or V; L16 replaced with A, G, I, S, T, M, or V; K17 replaced with H, or R; K18 replaced with H, or R; R19 replaced with H, or K; E20 replaced with D; E21 replaced with D; M22 replaced with A, G, I, L, S, T, or V; K23 replaced with H, or R; L24 replaced with A, G, I, S, T, M, or V; K25 replaced with H, or R; E26 replaced with D; V28 replaced with A, G, I, L, S, T, or M; S29 replaced with A, G, I, L, T, M, or V; I30 replaced with A, G, L, S, T, M, or V; L31 replaced with A, G, I, S, T, M, or V; R33 replaced with H, or K; K34 replaced with H, or R; E35 replaced with D; S36 replaced with A, G, I, L, T, M, or V; S38 replaced with A, G, I, L, T, M, or V; V39 replaced with A, G, I, L, S, T, or M; R40 replaced with H, or K; S41 replaced with A, G, I, L, T, M, or V; S42 replaced with A, G, I, L, T, M, or V; K43 replaced with H, or R; D44 replaced with E; G45 replaced with A, I, L, S, T, M, or V; K46 replaced with H, or R; L47 replaced with A, G, I, S, T, M, or V; L48 replaced with A, G, I, S, T, M, or V; A49 replaced with G, I, L, S, T, M, or V; A50 replaced with G, I, L, S, T, M, or V; T51 replaced with A, G, I, L, S, M, or V; L52 replaced with A, G, I, S, T, M, or V; L53 replaced with A, G, I, S, T, M, or V; L54 replaced with A, G, I, S, T, M, or V; A55 replaced with G, I, L, S, T, M, or V; L56 replaced with A, G, I, S, T, M, or V; L57 replaced with A, G, I, S, T, M, or V; S58 replaced with A, G, I, L, T, M, or V; L61 replaced with A, G, I, S, T, M, or V; T62 replaced with A, G, I, L, S, M, or V; V63 replaced with A, G, I, L, S, T, or M; V64 replaced with A, G, I, L, S, T, or M; S65 replaced with A, G, I, L, T, M, or V; F66 replaced with W, or Y; Y67 replaced with F, or W; Q68 replaced with N; V69 replaced with A, G, I, L, S, T, or M; A70 replaced with G, I, L, S, T, M, or V; A71 replaced with G, I, L, S, T, M, or V; L72 replaced with A, G, I, S, T, M, or V; Q73 replaced with N; G74 replaced with A, I, L, S, T, M, or V; D75 replaced with E; L76 replaced with A, G, I, S, T, M, or V; A77 replaced with G, I, L, S, T, M, or V; S78 replaced with A, G, I, L, T, M, or V; L79 replaced with A, G, I, S, T, M, or V; R80 replaced with H, or K; A81 replaced with G, I, L, S, T, M, or V; E82 replaced with D; L83 replaced with A, G, I, S, T, M, or V; Q84 replaced with N; G85 replaced with A, I, L, S, T, M, or V; H86 replaced with K, or R; H87 replaced with K, or R; A88 replaced with G, I, L, S, T, M, or V; E89 replaced with D; K90 replaced with H, or R; L91 replaced with A, G, I, S, T, M, or V; A93 replaced with G, I, L, S, T, M, or V; G94 replaced with A, I, L, S, T, M, or V; A95 replaced with G, I, L, S, T, M, or V; G96 replaced with A, I, L, S, T, M, or V; A97 replaced with G, I, L, S, T, M, or V; K99 replaced with H, or R; A100 replaced with G, I, L, S, T, M, or V; G101 replaced with A, I, L, S, T, M, or V; L102 replaced with A, G, I, S, T, M, or V; E103 replaced with D; E104 replaced with D; A105 replaced with G, I, L, S, T, M, or V; A107 replaced with G, I, L, S, T, M, or V; V108 replaced with A, G, I, L, S, T, or M; T109 replaced with A, G, I, L, S, M, or V; A110 replaced with G, I, L, S, T, M, or V; G111 replaced with A, I, L, S, T, M, or V; L112 replaced with A, G, I, S, T, M, or V; K113 replaced with H, or R; I114 replaced with A, G, L, S, T, M, or V; F115 replaced with W, or Y; E116 replaced with D; A119 replaced with G, I, L, S, T, M, or V; G121 replaced with A, I, L, S, T, M, or V; E122 replaced with D; G123 replaced with A, I, L, S, T, M, or V; N124 replaced with Q; S125 replaced with A, G, I, L, T, M, or V; S126 replaced with A, G, I, L, T, M, or V; Q127 replaced with N; N128 replaced with Q; S129 replaced with A, G, I, L, T, M, or V; R130 replaced with H, or K; N131 replaced with Q; K132 replaced with H, or R; R133 replaced with H, or K; A134 replaced with G, I, L, S, T, M, or V; V135 replaced with A, G, I, L, S, T, or M; Q136 replaced with N; G137 replaced with A, I, L, S, T, M, or V; E139 replaced with D; E140 replaced with D; T141 replaced with A, G, I, L, S, M, or V; G142 replaced with A, I, L, S, T, M, or V; S143 replaced with A, G, I, L, T, M, or V; Y144 replaced with F, or W; T145 replaced with A, G, I, L, S, M, or V; F146 replaced with W, or Y; V147 replaced with A, G, I, L, S, T, or M; W149 replaced with F, or Y; L150 replaced with A, G, I, S, T, M, or V; L151 replaced with A, G, I, S, T, M, or V; S152 replaced with A, G, I, L, T, M, or V; F153 replaced with W, or Y; K154 replaced with H, or R; R155 replaced with H, or K; G156 replaced with A, I, L, S, T, M, or V; S157 replaced with A, G, I, L, T, M, or V; A158 replaced with G, I, L, S, T, M, or V; L159 replaced with A, G, I, S, T, M, or V; E160 replaced with D; E161 replaced with D; K162 replaced with H, or R; E163 replaced with D; N164 replaced with Q; K165 replaced with H, or R; I166 replaced with A, G, L, S, T, M, or V; L167 replaced with A, G, I, S, T, M, or V; V168 replaced with A, G, I, L, S, T, or M; K169 replaced with H, or R; E170 replaced with D; T171 replaced with A, G, I, L, S, M, or V; G172 replaced with A, I, L, S, T, M, or V; Y173 replaced with F, or W; F174 replaced with W, or Y; F175 replaced with W, or Y; I176 replaced with A, G, L, S, T, M, or V; Y177 replaced with F, or W; G178 replaced with A, I, L, S, T, M, or V; Q179 replaced with N; V180 replaced with A, G, I, L, S, T, or M; L181 replaced with A, G, I, S, T, M, or V; Y182 replaced with F, or W; T183 replaced with A, G, I, L, S, M, or V; D184 replaced with E; K185 replaced with H, or R; T186 replaced with A, G, I, L, S, M, or V; Y187 replaced with F, or W; A188 replaced with G, I, L, S, T, M, or V; M189 replaced with A, G, I, L, S, T, or V; G190 replaced with A, I, L, S, T, M, or V; H191 replaced with K, or R; L192 replaced with A, G, I, S, T, M, or V; I193 replaced with A, G, L, S, T, M, or V; Q194 replaced with N; R195 replaced with H, or K; K196 replaced with H, or R; K197 replaced with H, or R; V198 replaced with A, G, I, L, S, T, or M; H199 replaced with K, or R; V200 replaced with A, G, I, L, S, T, or M; F201 replaced with W, or Y; G202 replaced with A, I, L, S, T, M, or V; D203 replaced with E; E204 replaced with D; L205 replaced with A, G, I, S, T, M, or V; S206 replaced with A, G, I, L, T, M, or V; L207 replaced with A, G, I, S, T, M, or V; V208 replaced with A, G, I, L, S, T, or M; T209 replaced with A, G, I, L, S, M, or V; L210 replaced with A, G, I, S, T, M, or V; F211 replaced with W, or Y; R212 replaced with H, or K; I214 replaced with A, G, L, S, T, M, or V; Q215 replaced with N; N216 replaced with Q; M217 replaced with A, G, I, L, S, T, or V; E219 replaced with D; T220 replaced with A, G, I, L, S, M, or V; L221 replaced with A, G, I, S, T, M, or V; N223 replaced with Q; N224 replaced with Q; S225 replaced with A, G, I, L, T, M, or V; Y227 replaced with F, or W; S228 replaced with A, G, I, L, T, M, or V; A229 replaced with G, I, L, S, T, M, or V; G230 replaced with A, I, L, S, T, M, or V; I231 replaced with A, G, L, S, T, M, or V; A232 replaced with G, I, L, S, T, M, or V; K233 replaced with H, or R; L234 replaced with A, G, I, S, T, M, or V; E235 replaced with D; E236 replaced with D; G237 replaced with A, I, L, S, T, M, or V; D238 replaced with E; E239 replaced with D; L240 replaced with A, G, I, S, T, M, or V; Q241 replaced with N; L242 replaced with A, G, I, S, T, M, or V; A243 replaced with G, I, L, S, T, M, or V; I244 replaced with A, G, L, S, T, M, or V; R246 replaced with H, or K; E247 replaced with D; N248 replaced with Q; A249 replaced with G, I, L, S, T, M, or V; Q250 replaced with N; I251 replaced with A, G, L, S, T, M, or V; S252 replaced with A, G, I, L, T, M, or V; L253 replaced with A, G, I, S, T, M, or V; D254 replaced with E; G255 replaced with A, I, L, S, T, M, or V; D256 replaced with E; V257 replaced with A, G, I, L, S, T, or M; T258 replaced with A, G, I, L, S, M, or V; F259 replaced with W, or Y; F260 replaced with W, or Y; G261 replaced with A, I, L, S, T, M, or V; A262 replaced with G, I, L, S, T, M, or V; L263 replaced with A, G, I, S, T, M, or V; K264 replaced with H, or R; L265 replaced with A, G, I, S, T, M, or V; and/or L266 replaced with A, G, I, S, T, M, or V.

In another embodiment, site directed changes at the amino acid level of B Lymphocyte Stimulator can be made by replacing a particular amino acid with a conservative substitution. Antibodies of the present invention may bind B Lymphocyte Stimulator amino acid sequences containing conservative substitution mutations of the polypeptide of any one of SEQ ID NOS:3230-3237.

Amino acids in the B Lymphocyte Stimulator polypeptides that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for functional activity, such ligand binding and the ability to stimulate lymphocyte (e.g., B cell) as, for example, proliferation, differentiation, and/or activation. Accordingly, antibodies of the present invention may bind amino acids in the B Lymphocyte Stimulator polypeptides that are essential for function. In preferred embodiments, antibodies of the present invention bind amino acids in the B Lymphocyte Stimulator polypeptides that are essential for function and inhibit B Lymphocyte Stimulator polypeptide function. In other preferred embodiments, antibodies of the present invention bind amino acids in the B Lymphocyte Stimulator polypeptides that are essential for function and enhance B Lymphocyte Stimulator polypeptide function.

Of special interest are substitutions of charged amino acids with other charged or neutral amino acids which may produce proteins with highly desirable improved characteristics, such as less aggregation. Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical formulations, because aggregates can be immunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).

In another embodiment, the invention provides for antibodies that bind polypeptides having amino acid sequences containing non-conservative substitutions of the amino acid sequence provided in SEQ ID NO:3228. For example, non-conservative substitutions of the B Lymphocyte Stimulator protein sequence provided in SEQ ID NO:3228 include: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D2 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D3 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E6 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R7 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E8 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q9 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S10 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R11 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L12 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C15 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K17 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K18 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R19 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E20 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E21 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M22 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K23 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L24 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K25 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E26 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C27 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V28 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S29 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I30 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L31 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P32 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R33 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K34 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E35 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S36 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P37 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R40 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S42 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K43 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D44 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G45 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K46 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L47 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T51 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L54 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A55 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L57 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S58 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C59 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; C60 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L61 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T62 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S65 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F66 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y67 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q68 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A71 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L72 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q73 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G74 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D75 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L76 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L79 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R80 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A81 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E82 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L83 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q84 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G85 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H86 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H87 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K90 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L91 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P92 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A93 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G94 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A95 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P98 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K99 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G101 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L102 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E103 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P106 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A107 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T109 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A110 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G111 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K113 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F115 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E116 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P117 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P118 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P120 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G121 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E122 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G123 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N124 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S126 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q127 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N128 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S129 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R130 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N131 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K132 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R133 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V135 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q136 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G137 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P138 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E139 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E140 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T143 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q144 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; D145 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C146 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L147 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q148 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L149 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D152 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S153 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E154 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T155 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T157 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q159 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K160 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G161 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S162 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y163 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T164 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F165 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V166 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P167 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W168 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L169 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S171 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F172 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K173 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R174 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G175 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A177 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E179 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E180 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K181 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E182 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N183 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K184 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L186 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V187 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K188 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E189 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G191 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y192 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F193 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F194 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I195 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y196 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G197 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q198 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V199 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y201 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D203 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K204 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y206 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G209 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H210 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L211 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I212 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q213 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R214 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K215 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K216 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H218 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V219 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F220 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D222 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E223 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L224 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L226 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V227 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T228 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F230 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R231 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C232 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; I233 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q234 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N235 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; M236 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P237 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E238 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T239 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P241 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; N242 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N243 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S244 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C245 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Y246 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S247 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A248 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G249 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I250 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A251 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K252 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E254 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E255 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G256 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D257 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E258 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L259 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q260 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L261 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A262 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I263 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P264 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R265 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E266 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N267 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A268 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q269 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I270 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S271 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L272 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D273 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G274 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D275 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V276 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T277 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F278 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F279 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G280 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L282 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K283 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L284 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L285 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.

In an additional embodiment, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides comprising, or alternatively consisting of, a B Lymphocyte Stimulator amino acid sequence in which more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and 50) is replaced with the substituted amino acids as described above (either conservative or nonconservative).

In another embodiment of the invention, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides with non-conservative substitutions of the sequence provided in SEQ ID NO:3229 including: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D2 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D3 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E6 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R7 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E8 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q9 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S10 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R11 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L12 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C15 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K17 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K18 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R19 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E20 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E21 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M22 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K23 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L24 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K25 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E26 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C27 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V28 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S29 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I30 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L31 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P32 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R33 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K34 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E35 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S36 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P37 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R40 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S42 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K43 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D44 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G45 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K46 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L47 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T51 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L54 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A55 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L57 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S58 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C59 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; C60 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L61 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T62 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S65 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F66 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y67 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q68 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A71 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L72 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q73 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G74 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D75 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L76 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L79 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R80 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A81 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E82 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L83 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q84 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G85 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H86 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H87 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K90 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L91 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P92 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A93 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G94 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A95 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P98 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K99 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G101 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L102 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E103 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P106 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A107 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T109 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A110 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G111 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K113 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F115 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E116 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P117 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P118 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P120 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G121 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E122 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G123 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N124 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S126 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q127 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N128 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S129 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R130 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N131 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K132 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R133 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V135 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q136 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G137 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P138 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E139 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E140 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S143 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y144 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T145 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F146 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V147 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P148 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W149 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S152 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F153 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K154 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R155 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G156 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S157 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L159 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E160 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E161 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K162 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E163 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N164 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K165 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I166 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L167 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V168 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K169 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E170 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T171 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G172 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y173 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F174 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F175 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y177 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q179 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V180 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y182 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T183 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D184 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K185 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T186 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y187 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A188 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M189 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H191 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L192 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I193 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q194 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R195 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K196 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K197 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V198 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H199 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F201 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D203 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E204 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S206 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T209 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L210 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F211 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R212 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C213 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; I214 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q215 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N216 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; M217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P218 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E219 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T220 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P222 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; N223 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N224 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C226 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Y227 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S228 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G230 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I231 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A232 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K233 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L234 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E235 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E236 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G237 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D238 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E239 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q241 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L242 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 1244 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P245 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R246 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E247 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N248 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A249 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q250 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I251 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S252 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D254 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G255 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D256 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V257 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T258 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F259 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F260 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G261 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A262 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L263 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K264 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L265 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.

In another embodiment, site directed changes at the amino acid level of B Lymphocyte Stimulator can be made by replacing a particular amino acid with a non-conservative substitution. Antibodies of the present invention may bind B Lymphocyte Stimulator amino acid sequences containing non-conservative substitution mutations of the polypeptide of any one of SEQ ID NOS:3230-3237.

In an additional embodiment, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides comprising, or alternatively consisting of, a B Lymphocyte Stimulator amino acid sequence in which more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and 50) is replaced with the substituted amino acids as described above (either conservative or nonconservative).

Replacement of amino acids can also change the selectivity of the binding of a ligand to cell surface receptors. For example, Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors. Since B Lymphocyte Stimulator is a member of the TNF polypeptide family, mutations similar to those in TNF-alpha are likely to have similar effects in B Lymphocyte Stimulator polypeptides.

Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

Since B Lymphocyte Stimulator is a member of the TNF-related protein family, mutations may be made in sequences encoding amino acids in the TNF conserved domain, e.g., in positions Gly-191 through Leu-284 of SEQ ID NO:3228 or in positions Gly-172 through Leu-265 of SEQ ID NO:3229, may modulate rather than completely eliminate functional activities (e.g., biological activities) of B Lymphocyte Stimulator polypeptides or fragments or variants thereof. Accordingly, antibodies of the present invention may bind B Lymphocyte Stimulator polypeptides that have mutations in the TNF conserved domain. In preferred embodiments, antibodies of the present invention may bind B Lymphocyte Stimulator polypeptides that have mutations in the TNF conserved domain and act as antagonists of B Lymphocyte Stimulator. In other preferred embodiments, antibodies of the present invention may bind B Lymphocyte Stimulator polypeptides that have mutations in the TNF conserved domain and act as agonists of B Lymphocyte Stimulator.

Recombinant DNA technology known to those skilled in the art (see, for instance, DNA shuffling supra) can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.

Thus, the invention also encompasses antibodies that bind B Lymphocyte Stimulator derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate B Lymphocyte Stimulator polypeptides, e.g., that are better suited for expression, scale up, etc., in the host cells. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites. To this end, a variety of amino acid substitutions at one or both of the first or third amino acid positions on any one or more of the glycosylation recognition sequences in the B Lymphocyte Stimulator polypeptides of the invention, and/or an amino acid deletion at the second position of any one or more such recognition sequences will prevent glycosylation of the B Lymphocyte Stimulator at the modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5(6):1193-1197). By way of non-limiting example, mutation of the serine at position 244 to alanine either singly or in combination with mutation of the asparagine at position 242 to glutamine abolishes glycosylation of the mature soluble form of B Lymphocyte Stimulator (e.g., amino acids 134-285 of SEQ ID NO:3228) when expressed in the yeast Pichea pastoris. A mutant B Lymphocyte Stimulator polypeptide in which only the asparagine at position 242 is mutated to glutamine, is still gycosylated when expressed in Pichea pastoris. In this mutant, the glycosylation event may be due to the activation or unmasking of an O-linked glyscosylation site at serine 244. Similar mutations affecting glycosylation could also be made in the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3229, i.e., aspargine-223 to glutamine and/or serine-224 to alanine of SEQ ID NO:3229. Additionally, one or more of the amino acid residues of the polypeptides of the invention (e.g., arginine and lysine residues) may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins. One possible result of such a mutation is that B Lymphocyte Stimulator polypeptide of the invention is not cleaved and released from the cell surface. Accordingly, antibodies of the invention may bind B Lymphocyte Stimulator derivatives and analogs that have one or more amino acid residues deleted, added, or substituted. In other embodiments, antibodies of the invention may bind B Lymphocyte Stimulator derivatives, variants or analogs that are unable to be cleaved from the cell surface.

In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Lys-132 and/or Arg-133 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, to prevent or diminish release of the soluble form of B Lymphocyte Stimulator from cells expressing B Lymphocyte Stimulator. In a more specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Lys-132 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to Ala-132. In another, nonexclusive specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Arg-133 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to Ala-133. These mutated proteins have uses such as, for example, in ex vivo therapy or gene therapy, to engineer cells expressing a B Lymphocyte Stimulator polypeptide that is retained on the surface of the engineered cells.

In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-146 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomerization of the mutant B Lymphocyte Stimulator polypeptide when expressed in an expression system. In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-146 is replaced with a serine amino acid residue.

In another specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-232 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomerization of the mutant B Lymphocyte Stimulator polypeptide when expressed in an expression system. In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-232 is replaced with a serine amino acid residue. Polypeptides encoding these polypeptides are also encompassed by the invention.

In yet another specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-245 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomerization of the mutant B Lymphocyte Stimulator polypeptide when expressed in an expression system. In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-245 is replaced with a serine amino acid residue. Polypeptides encoding these polypeptides are also encompassed by the invention.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of the B Lymphocyte Stimulator polypeptides can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).

The antibodies of the present invention bind B Lymphocyte Stimulator polypeptides including the complete polypeptide encoded by the deposited cDNA (ATCC™ Deposit No. 97768) including the intracellular, transmembrane and extracellular domains of the polypeptide encoded by the deposited cDNA, the mature soluble polypeptide encoded by the deposited cDNA, the extracellular domain minus the intracellular and transmembrane domains of the protein, the complete polypeptide of SEQ ID NO:3228, the mature soluble polypeptide of SEQ ID NO:3228, e.g., amino acids 134-285 of SEQ ID NO:3228, the extracellular domain of SEQ ID NO:3228, amino acid residues 73-285 of SEQ ID NO:3228 minus the intracellular and transmembrane domains, as well as polypeptides which have at least 80%, 85%, 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The antibodies of the present invention bind B Lymphocyte Stimulator polypeptides including the complete polypeptide encoded by the deposited cDNA including the intracellular, transmembrane and extracellular domains of the polypeptide encoded by the deposited cDNA (ATCC™ Deposit No. 203518), the mature soluble polypeptide encoded by the deposited cDNA, the extracellular domain minus the intracellular and transmembrane domains of the protein, the complete polypeptide of SEQ ID NO:3229, the mature soluble of SEQ ID NO:3229, e.g., amino acid residues 134-266 of SEQ ID NO:3229, the extracellular domain of SEQ ID NO:3229, e.g., amino acid residues 73-266 of SEQ ID NO:3229 minus the intracellular and transmembrane domains, as well as polypeptides which have at least 80%, 85%, 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Further antibodies of the present invention bind polypeptides including polypeptides at least 80%, or at least 85% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA (ATCC™ Deposit No. 97768) or to the polypeptide of SEQ ID NO:3228, and also include antibodies that bind portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.

Further antibodies of the present invention bind polypeptides including polypeptides at least 80%, or at least 85% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA (ATCC™ Deposit No. 203518) or to the polypeptide of SEQ ID NO:3229, and also include antibodies that bind portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. Polynucleotides encoding these polypeptides are also encompassed by the invention.

By “% similarity” for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a reference amino acid sequence of a B Lymphocyte Stimulator polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the B Lymphocyte Stimulator polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of SEQ ID NO:3228, the amino acid sequence encoded by the deposited cDNA clone HNEDU15 (ATCC™ Accession No. 97768), or fragments thereof, or, for instance, to the amino acid sequence of SEQ ID NO:3229, the amino acid sequence encoded by the deposited cDNA clone HDPMC52 (ATCC™ Accession No. 203518), or fragments thereof, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.

Antibodies that Immunospecifically Bind B Lymphocyte Stimulator Polypeptides

The present invention also encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator polypeptides, which antibodies comprise, or alternatively consist of, all or a portion of a heavy and/or light chain variable domain of the scFvs referred to in Table 1.

The present invention also encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor function in an animal, preferably a mammal, and most preferably a human, comprising using antibodies (including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator. Diseases and disorders which can be detected, diagnosed or prognosed with the antibodies of the invention include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).

The present invention further encompasses methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor function in an animal, preferably a mammal, and most preferably a human, comprising administering to said animal an effective amount of one or more antibodies (including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator. Diseases and disorders which can be prevented, treated or inhibited by administering an effective amount of one or more antibodies or molecules of the invention include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).

Anti-B Lymphocyte Stimulator Antibodies

The antibodies of the present invention were discovered, in part, using phage display technology. Single chain antibody molecules (“scFvs”) displayed on the surface of phage particles were screened to identify those scFvs that immunospecifically bind to B Lymphocyte Stimulator, including the membrane-bound form and soluble form of B Lymphocyte Stimulator. The present invention encompasses the scFvs and portions thereof that were identified to immunospecifically bind to B Lymphocyte Stimulator, including scFvs that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, scFvs that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, and scFvs that immunospecifically bind to both the soluble form and membrane-bound form of B Lymphocyte Stimulator. In particular, the present invention encompasses scFvs comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NOS: 1-2128, as referred to in Table 1. Preferably, the scFvs of the present invention comprise, or alternatively consist of, the amino acid sequence of SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908. The scFvs include scFvs that bind to soluble B Lymphocyte Stimulator (e.g., scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1563-1880), scFvs that bind to the membrane-bound form of B Lymphocyte Stimulator (e.g., scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1881-2128), and scFvs that bind to both the soluble form and the membrane-bound form of B Lymphocyte Stimulator (e.g., scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1-1562). Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.

In one embodiment of the present invention, scFvs that immunospecifically bind to B Lymphocyte Stimulator comprise a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1 and/or any one of the VL domains referred to in Table 1. In preferred embodiments, scFvs of the present invention comprise the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention comprise the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In another embodiment, scFvs that immunospecifically bind to B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs referred to in Table 1 and/or any one, two, three, or more of the VL CDRs referred to in Table 1. In preferred embodiments, scFvs of the present invention comprise the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention comprise the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. Molecules comprising, or alternatively consisting of, antibody fragments or variants of the scFvs referred to in Table 1 that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.

(Table 1 can be found at the end of the specification just prior to the claims.)

In another embodiment of the present invention, an scFv that immunospecifically binds to a soluble form of B Lymphocyte Stimulator, comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1563-1880 as referred to in Table 1. In a preferred embodiment, an scFv that immunospecifically binds to a soluble form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1570-1595. In an even more preferred embodiment, an scFv that immunospecifically binds to a soluble form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1563-1569.

In another embodiment of the present invention, an scFv that immunospecifically binds to a membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1881-2128 as referred to in Table 1. In a preferred embodiment, an scFv that immunospecifically binds to a membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1886-1908. In an even more preferred embodiment, an scFv that immunospecifically binds to a membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1881-1885.

In another embodiment of the present invention, an scFv that immunospecifically binds to both the soluble form and membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1-1562 as referred to in Table 1. In a preferred embodiment, an scFv that immunospecifically binds to both the soluble form and membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:834-872. In another preferred embodiment, an scFv that immunospecifically binds to both the soluble form and membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, any one of the amino acids sequences of SEQ ID NOS:1-46 or 321-329. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to the soluble form of B Lymphocyte Stimulator and/or the membrane-bound form of B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.

In another embodiment of the present invention, scFvs that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH domains contained in SEQ ID NOS:1563-1880 as disclosed in Table 1 and/or any one of the VL domains contained in SEQ ID NOS:1563-1880 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. In another embodiment, scFvs that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs SEQ ID NOS:1563-1880 as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs contained in contained SEQ ID NOS:1563-1880, as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the of the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In a preferred embodiment, scFvs that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s contained in SEQ ID NOS:1563-1880 as disclosed in Table 1 and/or any one of the VL CDR3s contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to B Lymphocyte Stimulator, preferably the soluble form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.

In another embodiment of the present invention, scFvs that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator comprise a polypeptide having the amino acid sequence of any one of the VH domains contained in SEQ ID NOS:1881-2128 as disclosed in Table 1 and/or any one of the VL domains contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In another embodiment, scFvs that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In a preferred embodiment, scFvs that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or any one of the VL CDR3s contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to B Lymphocyte Stimulator, preferably the membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.

In another embodiment of the present invention, scFvs that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH domains contained in SEQ ID NOS:1-1562 as disclosed in Table 1 and/or any one of the VL domains contained in SEQ ID NOS:1-1562 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In another embodiment, scFvs that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in SEQ ID NOS:1-1562 as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs contained in SEQ ID NOS:1-1562 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In a preferred embodiment, scFvs that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s contained in SEQ ID NOS:1-1562 as disclosed in Table 1 and/or any one of the VL CDR3s contained in SEQ ID NOS:1-1562, as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs or molecules, that immunospecifically bind to B Lymphocyte Stimulator, preferably the soluble and membrane-bound forms of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.

The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator. In particular, the invention provides antibodies corresponding to the scFvs referred to in Table 1, such scFvs may routinely be “converted” to immunoglobulin molecules by inserting, for example, the nucleotide sequences encoding the VH and/or VL domains of the scFv into an expression vector containing the constant domain sequences and engineered to direct the expression of the immunoglobulin molecule, as described in more detail in Example 20, infra.

In one embodiment, the invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one of the VH domains contained in the sequences referred to in Table 1. The present invention also provides antibodies that immunospecifically bind to a polypeptide, or polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one, two, three, or more of the VH CDRs contained in the sequences referred to in Table 1. Molecules comprising, or alternatively consisting of, these antibodies, or antibody fragments or variants thereof, that immunospecifically bind to B Lymphocyte Stimulator or a B Lymphocyte Stimulator fragment are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments and/or variants.

In one embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR referred to in Table 1. In particular, the invention provides antibodies that immunospecifically bind B Lymphocyte Stimulator, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VH CDR1 contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR2 contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1. In a preferred embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1. In yet another embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR1 contained in SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1; a VH CDR2 contained in SEQ ID NOS: SEQ ID NOS: SEQ ID NOS:834-872, 1570-1595, or 1886-1908; and/or a VH CDR3 contained in SEQ ID NOS: SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1. Preferably, antibodies of the invention comprise, or alternatively consist of, VH CDRs that are derived from the same scFv as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.

The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) that immunospecifically bind to a polypeptide, or polypeptide fragment of B Lymphocyte Stimulator. In particular, the invention provides antibodies wherein said antibodies comprise, or alternatively consist of, a VL domain having an amino acid sequence of any one of the VL domains referred to in Table 1. The present invention also provides antibodies that immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a VL CDR having an amino acid sequence of any one, two, three, or more of the VL CDRs contained in the sequences referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.

In one embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR referred to in Table 1. In particular, the invention provides antibodies that immunospecifically bind B Lymphocyte Stimulator, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VL CDR1 contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR2 contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1. In a preferred embodiment, antibodies comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID NOS: in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 disclosed in Table 1. In yet another embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator comprise, or alternatively consist of: a polypeptide having the amino acid sequence of a VL CDR1 contained in SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1; a VL CDR2SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1; and a VL CDR3 contained SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1. Preferably, antibodies of the invention comprise, or alternatively consist of, VL CDRs that are derived from the same scFv as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.

The present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a VH domain of one of the scFvs referred to in Table 1 combined with a VL domain of one of the scFvs referred to in Table 1, or other VL domain. The present invention further provides antibodies (including molecules comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a VL domain of one of the scFvs referred to in Table 1 combined with a VH domain of one of the scFvs referred to in Table 1, or other VH domain. In a preferred embodiment, antibodies that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH domain contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1 and a VL domain contained in contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1. In a further preferred embodiment, the antibodies of the invention comprise, or alternatively consist of, a VH and a VL domain from the same scFv as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.

The present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) that immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, one, two, three, or more VH CDRs and one, two, three or more VL CDRs, as referred to in Table 1. In particular, the invention provides for antibodies that immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a VH CDR1 and a VL CDR1, a VH CDR1 and a VL CDR2, a VH CDR1 and a VL CDR3, a VH CDR2 and a VL CDR1, VH CDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and a VH CDR1, a VH CDR3 and a VL CDR2, a VH CDR3 and a VL CDR3, or any combination thereof, of the VH CDRs and VL CDRs referred to in Table 1. In a preferred embodiment, one or more of these combinations are from the same scFv as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.

In a preferred embodiment the invention provides antibodies wherein the VH CDRX (where X=1, 2, or 3) and VL CDRY (where Y=1, 2, or 3) are from scFvs with the same specificity (i.e., from scFvs that bind soluble B Lymphocyte Stimulator, from scFvs that bind membrane-bound B Lymphocyte Stimulator, or from scFvs that bind both soluble and membrane-bound B Lymphocyte Stimulator. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.

The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term “antibody” encompasses not only whole antibody molecules, but also antibody fragments, as well as variants (including derivatives) of antibodies and antibody fragments. Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, single chain Fvs (scFvs), Fab fragments, F(ab′)₂ fragments, Fd fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass of immunoglobulin molecule. The antibodies of the present invention also include molecules comprising, or alternatively consisting of, a polypeptide having an amino acid sequence of a portion of an amino acid sequence contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908. Preferably, an antibody of the invention comprises, or alternatively consists of, a polypeptide having an amino acid sequence of a VH domain, VH CDR, VL domain, or VL CDR of any one those contained in the sequences referred to in Table 1. Antibodies of the invention also include molecules comprising, or alternatively consisting of, fragments or variants of the above antibodies that immunospecifically bind B Lymphocyte Stimulator.

Most preferably the antibodies of the present invention are whole antibodies or antibody fragments that immunospecifically bind human B Lymphocyte Stimulator. Antibody fragments of the invention that immunospecifically bind human B Lymphocyte Stimulator include, but are not limited to, Fab, Fab′ and F(ab′)₂, Fd fragments, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFvs), fragments comprising, or alternatively consisting of, either a VL or VH domain, and epitope binding fragments of any of the above.

B Lymphocyte Stimulator-binding antibody fragments, including single-chain antibodies, may comprise, or alternatively consist of, the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. In a preferred embodiment, the antibodies of the invention comprise, or alternatively consist of, a polypeptide that immunospecifically binds to B Lymphocyte Stimulator, said polypeptides comprise, or alternatively consist of, one, two, three, four, five, six or more CDRs referred to in Table 1, preferably a polypeptide having an amino acid sequence of a VH CDR3 and/or a VL CDR3 of contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1. Most preferably, antibodies of the invention comprise, or alternatively consist of, one, two, three, four, five, six or more CDRs from the same scFv, as referred to in Table 1. The antibodies of the invention may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Most preferably, the antibodies are human antibodies. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries and xenomice or other organisms that have been genetically engineered to produce human antibodies. For a detailed discussion of a few of the technologies for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; and Lonberg and Huszar, Int. Rev. Immunol 13:65-93 (1995), which are incorporated by reference herein in their entirety. Human antibodies or “humanized” chimeric monoclonal antibodies can be produced using techniques described herein or otherwise known in the art. For example, methods for producing chimeric antibodies are known in the art. See, for review the following references which are hereby incorporated in their entirety: Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985). In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

The antibodies of the present invention may be monovalent, bivalent, trivalent or multivalent. For example, monovalent scFvs can be multimerized either chemically or by association with another protein or substance. An scFv that is fused to a hexahistidine tag or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using anti-Flag antibodies (Stratagene, Inc.).

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a B Lymphocyte Stimulator polypeptide, or fragment thereof, or may be specific for both a B Lymphocyte Stimulator polypeptide, or fragment thereof, and a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et at., J. Immunol. 148:1547-1553 (1992).

The antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may bind immunospecifically to murine B Lymphocyte Stimulator (e.g., a polypeptide having the amino acid sequence of human B Lymphocyte Stimulator (SEQ ID NOS:3228 and/or 3229) or B Lymphocyte Stimulator expressed on human monocytes; murine B Lymphocyte Stimulator (SEQ ID NOS:3230 and/or 3231) or B Lymphocyte Stimulator expressed on murine monocytes; rat B Lymphocyte Stimulator (either the soluble forms as given in SEQ ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey B Lymphocyte Stimulator (e.g., the monkey B Lymphocyte Stimulator polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey B Lymphocyte Stimulator, or B Lymphocyte Stimulator expressed on monkey monocytes), preferably the antibodies of the invention bind immunospecifically to human B Lymphocyte Stimulator. Preferably, the antibodies of the invention bind immunospecifically to human and monkey B Lymphocyte Stimulator. Also preferably, the antibodies of the invention bind immunospecifically to human B Lymphocyte Stimulator and murine B Lymphocyte Stimulator. More preferably, antibodies of the invention, bind immunospecifically and with higher affinity to human B Lymphocyte Stimulator than to murine B Lymphocyte Stimulator.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, antibodies of the present invention cross react with APRIL (SEQ ID NO:3239; GenBank Accession No. AF046888; J. Exp. Med. 188(6):1185-1190; PCT International Publication WO97/33902). In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under hybridization conditions (as described herein).

In a specific embodiment, antibodies of the present invention cross react with APRIL (SEQ ID NO:3239; GenBank Accession No. AF046888; J. Exp. Med. 188(6):1185-1190; PCT International Publication WO97/33902). In specific embodiments, antibodies that immunospecifically bind both B Lymphocyte Stimulator and APRIL comprise all or a portion the BAB2001, BAB2080, BAB2015, BAB2019, BAB2087, BAB2016, BAB2034 or BAB2065 scFVs (SEQ ID NOS:3240-3247). These scFvs were isolated by panning a phage scFv library comprising VH and VL domains obtained from human bone marrow B cells (BM library). Phage from the BM phage library were first selected for binding to soluble B Lymphocyte Stimulator (amino acids 134-285 of SEQ ID NO:3228). A second round of selection for binding to the soluble form of APRIL (amino acids 105-250 of SEQ ID NO:3239) was then performed on the B Lymphocyte Stimulator binding phage selected in round one. A third round of selection for binding to the soluble form of APRIL (amino acids 105-250 of SEQ ID NO:3239) was then performed on the phage selected in round two. A final (fourth) round of selection for binding to the soluble form of B Lymphocyte Stimulator (amino acids 134-285 of SEQ ID NO:3228) was then performed on the phage selected in round three. Phage clones that bound B Lymphocyte Stimulator in the fourth round of selection were eluted with either 0.1M triethylamine (TEA) or with a TACI-Fc fusion protein (e.g., the extracellular domain of TACI (amino acids 31 to 159 of Genbank Accession No. AAC51790) fused to Fc). Eluted Phage were collected and sequenced (SEQ ID NOS:3240-3247). Of 79 sequences, there were 8 unique sequences (SEQ ID NOS:3240-3247).

Isolated scFv clones (e.g., scFvs corresponding to SEQ ID NOS:3240-3247 or other scFvs described in Table 1) or antibodies comprising at least a portion of said scFV clones may be screened for their ability to bind their ability to bind the soluble form of B Lymphocyte Stimulator and the soluble form of APRIL by ELISA. Isolated scFv clones or antibodies comprising at least a portion of said scFv clones may also be screened for their ability to inhibit binding of a soluble form of B Lymphocyte Stimulator or B Lymphocyte Stimulator heterotrimer to TACI, BCMA or BAFF-R (Genbank Accession Nos. AAC51790, NP_(—)00183, and NP_(—)443177, respectively). Isolated scFv clones or antibodies comprising at least a portion of said scFV clones may also be screened for their ability to inhibit B Lymphocyte Stimulator or B Lymphocyte Stimulator heterotrimer mediated biological activities (e.g. stimulation of B cell proliferation and/or stimulation of immunoglobulin production).

In specific embodiments, antibodies that immunospecifically bind both B Lymphocyte Stimulator and APRIL comprise all or a portion (e.g., VHCDR, VLCDR, VH domain, VL domain) of the BAB2001, BAB2015, BAB2016, BAB2019, BAB2034, BAB2065, or BAB2080 scFVs (SEQ ID NOS:3240-3247).

In preferred embodiments, the antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), immunospecifically bind to B Lymphocyte Stimulator and do not cross-react with any other antigens. In more preferred embodiments, the antibodies of the invention immunopecifically bind to B Lymphocyte Stimulator and do not cross-react with TRAIL, APRIL, Endokine-alpha, TNF-alpha, TNF-beta, Fas-L or LIGHT.

The present invention also provides for a nucleic acid molecule, generally isolated, encoding an antibody of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). In one embodiment, a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains referred to in Table 1. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR1 having an amino acid sequence of any one of the VH CDR1s referred to in Table 1. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR2 having an amino acid sequence of any one of the VH CDR2s referred to in Table 1. In yet another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR3 having an amino acid sequence of any one of the VH CDR3s referred to in Table 1. Nucleic acid molecules encoding antibodies that immunospecifically bind B Lymphocyte Stimulator and comprise, or alternatively consist of, fragments or variants of the VH domains and/or VH CDRs are also encompassed by the invention.

In another embodiment, a nucleic acid molecule of the invention encodes an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), comprising, or alternatively consisting of, a VL domain having an amino acid sequence of any one of the VL domains referred to in Table 1. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR1 having amino acid sequence of any one of the VL CDR1s referred to in Table 1. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR2 having an amino acid sequence of any one of the VL CDR2s referred to in Table 1. In yet another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR3 having an amino acid sequence of any one of the VL CDR3s referred to in Table 1. Nucleic acid encoding antibodies that immunospecifically bind B Lymphocyte Stimulator and comprise, or alternatively consist of, fragments or variants of the VL domains and/or VLCDR(s) are also encompassed by the invention.

In another embodiment, a nucleic acid molecule of the invention encodes an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains referred to in Table 1 and a VL domain having an amino acid sequence of any one of the VL domains referred to in Table 1. In another embodiment, a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH CDR1, a VL CDR1, a VH CDR2, a VL CDR2, a VH CDR3, a VL CDR3, or any combination thereof having an amino acid sequence referred to in Table 1. Nucleic acid encoding antibodies that immunospecifically bind B Lymphocyte Stimulator and comprise, or alternatively consist of, fragments or variants of the VL and/or domains and/or VHCDR(s) and/or VLCDR(s) are also encompassed by the invention.

The present invention also provides antibodies that comprise, or alternatively consist of, variants (including derivatives) of the VH domains, VH CDRs, VL domains, and VL CDRs described herein, which antibodies immunospecifically bind to B Lymphocyte Stimulator. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3. In specific embodiments, the variants encode substitutions of VHCDR3. In a preferred embodiment, the variants have conservative amino acid substitutions at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind B Lymphocyte Stimulator). Following mutagenesis, the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecifically bind B Lymphocyte Stimulator) can be determined using techniques described herein or by routinely modifying techniques known in the art.

The antibodies of the invention include derivatives (i.e., variants) that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not affect the ability of the antibody to immunospecifically bind to B Lymphocyte Stimulator. For example, but not by way of limitation, derivatives of the invention include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

In a specific embodiment, an antibody of the invention (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that immunospecifically binds B Lymphocyte Stimulator, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH or VL domains referred to in Table 1 under stringent conditions, e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C., or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3). In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH CDRs or VL CDRs referred to in Table 1 under stringent conditions, e.g., hybridization under conditions as described above, or under other stringent hybridization conditions which are known to those of skill in the art. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH CDR3s referred to in Table 1 under stringent conditions e.g., hybridization under conditions as described above, or under other stringent hybridization conditions which are known to those of skill in the art. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

In another embodiment, an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VH domains referred to in Table 1. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VH CDRs referred to in Table 1. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of the VH CDR3s referred to in Table 1. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

In another embodiment, an antibody of the invention (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL domains referred to in Table 1. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL CDRs referred to in Table 1. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL CDR3s referred to in Table 1. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

Antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may also be described or specified in terms of their binding affinity for to B Lymphocyte Stimulator polypeptides or fragments or variants of B Lymphocyte Stimulator polypeptides (e.g., to the soluble form of B Lymphocyte Stimulator and/or membrane-bound form of B Lymphocyte Stimulator). In specific embodiments, antibodies of the invention bind B Lymphocyte Stimulator polypeptides, or fragments or variants thereof, with a dissociation constant or K_(D) of less than or equal to 5×10⁻²M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, or 10⁻⁵ M. More preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with a dissociation constant or K_(D) less than or equal to 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸ M, or 10⁻⁸ M. Even more preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with a dissociation constant or K_(D) less than or equal to 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. The invention encompasses antibodies that bind B Lymphocyte Stimulator polypeptides with a dissociation constant or K_(D) that is within any one of the ranges that are between each of the individual recited values.

In specific embodiments, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with an off rate (k_(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹. More preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with an off rate (k_(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹. The invention encompasses antibodies that bind B Lymphocyte Stimulator polypeptides with an off rate (k_(off)) that is within any one of the ranges that are between each of the individual recited values.

In other embodiments, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with an on rate (k_(on)) of greater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹ sec⁻¹. More preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with an on rate (k_(on)) greater than or equal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷M⁻¹ sec⁻¹. The invention encompasses antibodies that bind B Lymphocyte Stimulator polypeptides with on rate (k_(on)) that is within any one of the ranges that are between each of the individual recited values.

The invention also encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that have one or more of the same biological characteristics as one or more of the antibodies described herein. By “biological characteristics” is meant, the in vitro or in vivo activities or properties of the antibodies, such as, for example, the ability to bind to B Lymphocyte Stimulator (e.g., the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, the soluble form and membrane-bound form of B Lymphocyte Stimulator), and/or an antigenic and/or epitope region of B Lymphocyte Stimulator), the ability to substantially block B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP_(—)001183; and/or BAFF-R—GenBank accession number NP_(—)443177) binding, or the ability to block B Lymphocyte Stimulator mediated biological activity (e.g., stimulation of B cell proliferation and immunoglobulin production). Optionally, the antibodies of the invention will bind to the same epitope as at least one of the antibodies specifically referred to herein. Such epitope binding can be routinely determined using assays known in the art.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that neutralize B Lymphocyte Stimulator or a fragment thereof, said antibodies comprising, or alternatively consisting of, a portion (i.e., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv referred to in Table 1, more preferably having an amino acid sequence contained in SEQ ID NOS:834-872, 1570-1595, or 1886-1908, and even more preferably having an amino acid sequence contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1, or a fragment or variant thereof. By an antibody that “neutralizes B Lymphocyte Stimulator or a fragment thereof” is meant an antibody that diminishes or abolishes the ability of B Lymphocyte Stimulator to bind to its receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP_(—)001183; and/or BAFF-R—GenBank accession number NP_(—)443177) to stimulate B cell proliferation, to stimulate immunoglobulin secretion by B cells, and/or to stimulate the B Lymphocyte Stimulator receptor signalling cascade. In one embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR domain in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit (i.e., diminish or abolish) B Lymphocyte Stimulator-mediated B cell proliferation as determined by any method known in the art such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, 1886-1908, and even more preferably having an amino acid sequence SEQ ID NOS:1-46, 321-329, 1563-1569, 1881-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908, as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit (i.e., diminish or abolish) B Lymphocyte Stimulator-mediated stimulation of B cell survival as determined by any method known in the art such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, 1886-1908, and even more preferably having an amino acid sequence SEQ ID NOS:1-46, 321-329, 1563-1569, 1881-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell survival, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908, as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell survival, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell survival, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell survival, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit (i.e., diminish or abolish) B Lymphocyte Stimulator-mediated stimulation of B cell differentiation as determined by any method known in the art such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, 1886-1908, and even more preferably having an amino acid sequence SEQ ID NOS:1-46, 321-329, 1563-1569, 1881-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell differentiation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908, as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell differentiation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell differentiation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell differentiation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit (i.e., diminish or abolish) B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells as determined by any method known in the art such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, 1886-1908, and even more preferably having an amino acid sequence SEQ ID NOS:1-46, 321-329, 1563-1569, 1881-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908, as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that enhance the activity of B Lymphocyte Stimulator or a fragment thereof, said antibodies comprising, or alternatively consisting of, a portion (i.e., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, or 1886-1908, and preferably having an amino acid sequence of SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885, as disclosed in Table 1, or a fragment or variant thereof. By an antibody that “enhances the activity of B Lymphocyte Stimulator or a fragment thereof” is meant an antibody increases the ability of B Lymphocyte Stimulator to bind to its receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP_(—)001183; and/or BAFF-R—GenBank accession number NP_(—)443177), to stimulate B cell proliferation, to stimulate immunoglobulin secretion by B cells, and/or to stimulate the B Lymphocyte Stimulator receptor signalling cascade. In one embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that enhances B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that stimulate B Lymphocyte Stimulator-mediated B cell proliferation as determined by any method known in the art, such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence of SEQ ID NOS:834-872, 1570-1595, or 1886-1908, and even more preferably having an amino acid sequence of SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that stimulates B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that stimulates B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that stimulates B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that stimulates B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for fusion proteins comprising, or alternatively consisting of, an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that immunospecifically binds to B Lymphocyte Stimulator, and a heterologous polypeptide. Preferably, the heterologous polypeptide to which the antibody is fused to is useful for B-cell function or is useful to target the antibody to B-cells. In an alternative preferred embodiment, the heterologous polypeptide to which the antibody is fused to is useful for monocyte cell function or is useful to target the antibody to a monocyte. In another embodiment, the heterologous polypeptide to which the antibody is fused is albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-x of human serum albumin, where x is an integer from 1 to 585 and the albumin fragment has human serum albumin activity. In another preferred embodiment, antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide).

In one embodiment, a fusion protein of the invention comprises, or alternatively consists of, a polypeptide having the amino acid sequence of any one or more of the VH domains referred to in Table 1 or the amino acid sequence of any one or more of the VL domains referred to in Table 1 or fragments or variants thereof, and a heterologous polypeptide sequence. In another embodiment, a fusion protein of the present invention comprises, or alternatively consists of, a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs referred to in Table 1, or the amino acid sequence of any one, two, three, or more of the VL CDRs referred to in Table 1, or fragments or variants thereof, and a heterologous polypeptide sequence. In a preferred embodiment, the fusion protein comprises, or alternatively consists of, a polypeptide having the amino acid sequence of, a VH CDR3 referred to in Table 1, or fragment or variant thereof, and a heterologous polypeptide sequence, which fusion protein immunospecifically binds to B Lymphocyte Stimulator. In another embodiment, a fusion protein comprises, or alternatively consists of a polypeptide having the amino acid sequence of at least one VH domain referred to in Table 1 and the amino acid sequence of at least one VL domain referred to in Table 1 or fragments or variants thereof, and a heterologous polypeptide sequence. Preferably, the VH and VL domains of the fusion protein correspond to the same scFv referred to in Table 1. In yet another embodiment, a fusion protein of the invention comprises, or alternatively consists of a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs referred to in Table 1 and the amino acid sequence of any one, two, three or more of the VL CDRs referred to in Table 1, or fragments or variants thereof, and a heterologous polypeptide sequence. Preferably, two, three, four, five, six, or more of the VHCDR(s) or VLCDR(s) correspond to the same scFv referred to in Table 1. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.

The present invention also provides: antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that immunospecifically bind to the soluble form of B Lymphocyte Stimulator; antibodies that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator; and antibodies that immunospecifically bind to both the soluble form and membrane-bound form of B Lymphocyte Stimulator.

In one embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS:1563-1880 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1, or fragment(s) or variant(s) (including derivative) thereof. Preferably, the VH and VL domains of the antibody correspond to the same scFv as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained SEQ ID NOS: 1563-1880 as disclosed in Table 1 and/or the amino acid sequence of any one, two, three, or more of the VL CDRs contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, two, three, four, five, six or more of the VH and VL CDRs of the antibody correspond to the same scFv as disclosed in Table 1. In a preferred embodiment, antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, the VHCDR3 and VLCDR3 of the antibody correspond to the same scFv, as disclosed in Table 1. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

In another embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1, or a fragment or variant thereof. Preferably, the VH and VL domains of the antibody correspond to the same scFv as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or the amino acid sequence of any one, two, three, or more of the VL CDRs contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, two, three, four, five, six or more of the VH and VL CDRs of the antibody correspond to the same scFv as disclosed in Table 1. In a preferred embodiment, antibodies that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, the VHCDR3 and VLCDR3 of the antibody correspond to the same scFv, as disclosed in Table 1. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

In another embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator, are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS: 1-1562 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS: 1-1562 as disclosed in Table 1, or a fragment or variant thereof. Preferably, the VH and VL domains of the antibody correspond to the same scFv as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in SEQ ID NOS: 1-1562 as disclosed in Table 1 and/or the amino acid sequence of any one, two, three, or more of the VL CDRs contained in SEQ ID NOS: 1-1562 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, two, three, four, five, six or more of the VH and VL CDRs of the antibody correspond to the same scFv as disclosed in Table 1. In a preferred embodiment, antibodies that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS: 1-1562, disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS: 1-1562, disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, the VHCDR3 and VLCDR3 of the antibody correspond to the same scFv, as disclosed in Table 1.

The present invention also provides for mixtures of antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator, wherein the mixture has at least one, two, three, four, five or more different antibodies of the invention. In particular, the invention provides for mixtures of different antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the membrane-bound form and soluble form of B Lymphocyte Stimulator. In specific embodiments, the invention provides mixtures of at least 2, preferably at least 4, at least 6, at least 8, at least 10, at least 12, at least 15, at least 20, or at least 25 different antibodies that immunospecifically bind to B Lymphocyte Stimulator, wherein at least 1, at least 2, at least 4, at least 6, or at least 10, antibodies of the mixture is an antibody of the invention. In a specific embodiment, each antibody of the mixture is an antibody of the invention.

The present invention also provides for panels of antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator, wherein the panel has at least one, two, three, four, five or more different antibodies of the invention. In particular, the invention provides for panels of different antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the membrane-bound form and soluble form of B Lymphocyte Stimulator. In specific embodiments, the invention provides for panels of antibodies that have different affinities for B Lymphocyte Stimulator, different specificities for B Lymphocyte Stimulator, or different dissociation rates.

The invention provides panels of at least 10, preferably at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000, antibodies. Panels of antibodies can be used, for example, in 96 well plates for assays such as ELISAs.

The present invention further provides for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention). In one embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR1s contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR2s contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS:1563-1880, as disclosed in Table 1 or a variant thereof.

The present invention further provides for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention). In one embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR1s contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR2s contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS:1881-2128 as disclosed in Table 1 or a variant thereof.

The present invention further provides for compositions comprising, one or more antibodies (including scFvs, or molecules comprising, or alternatively consisting of antibody fragments or variants of the invention). In one embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS:1-1562 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR1s contained in SEQ ID NOS:1-1562 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR2s contained in SEQ ID NOS:1-1562 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS:1-1562 as disclosed in Table 1 or a variant thereof.

Other embodiments of the present invention providing for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention) are listed below. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternative consist of, a polypeptide having an amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR1s contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR2s contained SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof.

Other embodiments of the present invention providing for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention) are listed below. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR1s contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR2s SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof.

Other embodiments of the present invention providing for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention) are listed below. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS:1-1562 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR1s contained in SEQ ID NOS:1-1562 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR2s SEQ ID NOS:1-1562 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS:1-1562 as disclosed in Table 1, or a variant thereof.

In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH domains in disclosed in Table 1, or a variant thereof, and an amino acid sequence of any one or more of the VL domains disclosed in Table 1, or a variant thereof wherein the VH and VL domains are from scFvs with the same specificity (i.e., from scFvs that bind soluble B Lymphocyte Stimulator (SEQ ID NOS:1563-1880), from scFvs that bind membrane-bound B Lymphocyte Stimulator (SEQ ID 1881-2128), or from scFvs that bind both soluble and membrane-bound B Lymphocyte Stimulator (SEQ ID NOS:1-1562). In a preferred embodiment the invention provides antibodies wherein the VH CDRX (where X=1, 2, or 3) and VL CDRY (where Y=1, 2, or 3) are from scFvs with the same specificity (i.e., from scFvs that bind soluble B Lymphocyte Stimulator (SEQ ID NOS:1563-1880), from scFvs that bind membrane-bound B Lymphocyte Stimulator (SEQ ID NOS:1881-2128), or from scFvs that bind both soluble and membrane-bound B Lymphocyte Stimulator (SEQ ID NOS:1-1562). In yet another embodiment, a composition of the present invention comprises one or more fusion proteins.

As discussed in more detail below, a composition of the invention may be used either alone or in combination with other compositions. The antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the present invention) may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

Antibodies of the present invention (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the present invention) may be used, for example, but not limited to, to purify and detect B Lymphocyte Stimulator, and to target the polypeptides of the present invention to cells expressing membrane-bound B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of B Lymphocyte Stimulator in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

Methods Producing Antibodies

The antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention) can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

The single chain Fvs disclosed in Table 1 were generated using phage display methods known in the art. Furthermore, other scFvs that immunospecifically bind B Lymphocyte Stimulator may be generated using phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The DNA encoding the VH and VL domains are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of interest (i.e., B Lymphocyte Stimulator or a fragment thereof) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include, but are not limited to, those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et at., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/O1 134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; WO97/13844; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

ScFvs that immunospecifcally bind to both B Lymphocyte Stimulator and APRIL polypeptides (preferably to the mature soluble forms of each) may be obtained, for example, by sequential rounds of selection for binding to one or the other of B Lymphocyte Stimulator and APRIL polypeptides. Thus in one embodiment, the present invention provides for a method of selecting phage that express scFvs that immunospecifically bind both B Lymphocyte Stimulator polypeptides and APRIL polypeptides, comprising at least one round of phage selection for binding to B Lymphocyte Stimulator polypeptide and at least one round of phage selection for binding to APRIL polypeptide. Selection for B Lymphocyte Stimulator binding may either precede or follow selection for APRIL binding. More than one round of selection for binding to either B Lymphocyte Stimulator or APRIL may be conducted.

ScFvs that immunospecifcally bind to a heterotrimer comprising at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide may be, for example, obtained by sequential rounds of selection for binding to one or the other of B Lymphocyte Stimulator and APRIL polypeptide. Thus in one embodiment, the present invention provides for a method of selecting phage that express scFvs that immunospecifically bind a heterotrimer comprising at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide, comprising at least one round of phage selection for binding to B Lymphocyte Stimulator polypeptide and at least one round of phage selection for binding to APRIL polypeptide. Selection for B Lymphocyte Stimulator binding may either precede or follow selection for APRIL binding. More than one round of selection for binding to either B Lymphocyte Stimulator or APRIL may be conducted.

Alternatively, scFvs that immunospecifcally bind to a heterotrimer comprising at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide may be obtained, for example, by selecting scFVs that bind to a B Lymphocyte Stimulator heterotrimer. A B Lymphocyte Stimulator heterotrimer may contain, for example, one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides (2B Lymphocyte Stimulator:1APRIL heterotrimer). Other B Lymphocyte Stimulator heterotrimers may contain, for example, one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides (1B Lymphocyte Stimulator:2APRIL heterotrimer). Preferably, the heterotrimers comprising B Lymphocyte Stimulator and APRIL polypeptides contain the mature forms of both the B Lymphocyte Stimulator and APRIL polypeptides. ScFvs may be selected in one or more rounds of selection for binding only to the 2B

Lymphocyte Stimulator:1APRIL heterotrimer, or only to the 1B Lymphocyte Stimulator:2APRIL heterotrimer. Alternatively, scFVs that immunospecifcally bind heterotrimers comprising B Lymphocyte Stimulator and APRIL polypeptides may be obtained through sequential rounds of screening on individual forms of the B Lymphocyte Stimulator/APRIL heterotrimer in any order and/or on both forms of the heterotrimer simultaneously, or any combination thereof.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).

The characteristics of an antibody, such as its on-rate, off-rate and/or overall affinity may be altered using in vitro mutation and selection techniques. This process is well known in the art and is commonly referred to as in vitro affinity maturation of antibodies. Starting with a given antibody that binds a particular antigen with a certain affinity, one of skill in the art can engineer variants of that antibody and test the antibody variants for altered (usually improved) antigen binding characteristics. The amino acid sequence of the VH and VL regions of such antibody variants may be substantially from that of the starting or original antibody; for example, the an antibody variants may comprise the original VH or VL paired with a different (from the original) VL or VH region, respectively. Alternatively, the amino acid sequence of the VH and VL region of such antibody variants may be quite similar to that of the starting or original antibody; for example, an antibody variant may have only a few amino acid changes in the VH and/or VL region. It is common for one to engineer the mutations in CDR regions, and particularly in the VHCDR3 region. Examples of both types of in vitro antibody affinity maturation are described for example, Thompson et al., (1996) The Journal of Molecular Biology 256:77-88. Moreover, a review of phage display antibody technology may be found in Vaughan et al., (1998) Nature Biotechnology 16:535-539; both of these articles are herein incorporated by reference in their entireties. The scFvs of SEQ ID NOS:10-37 are CDR3 mutants derived from the scFv of SEQ ID NO:9 and scFvs of SEQ ID NOS:291-327 are CDR3 mutants derived from the scFv of SEQ ID NO:2.

To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. Preferably, the vectors for expressing the VH or VL domains comprise a promoter suitable to direct expression of the heavy and light chains in the chosen expression system, a secretion signal, a cloning site for the immunoglobulin variable domain, immunoglobulin constant domains, and a selection marker such as neomycin. The VH and VL domains may also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.

Cell lines that express antibodies that comprise the VH and VL domains of scFvs of the invention have been deposited with the American Type Culture Collection (“ATCC™”) on the dates listed in Table 2 and given the ATCC™ Deposit Numbers identified in Table 2. The ATCC™ is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC™ deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.

TABLE 2 Corre- ATCC ™ ATCC ™ sponding SEQ ID Deposit Deposit Cell Line scFv NO: Number Date NSO-B11-15 I050B11-15 24 PTA-3238 Mar. 27, 2001 NSO-anti-B Lym- I006D08 2 PTA-3239 Mar. 27, 2001 phocyte Stim- ulator-6D08-18 NSO- anti-B Lym- I116A01 327 PTA-3240 Mar. 27, 2001 phocyte Stim- ulator-116A01-60 IO26C04K I026C04-K 1563 PTA-3241 Mar. 27, 2001 IO50A12 I050A12 12 PTA-3242 Mar. 27, 2001 IO50-B11 I050B11 9 PTA-3243 Mar. 27, 2001

Accordingly, in one embodiment, the invention provides antibodies that comprise the VH and VL domains of scFvs of the invention.

In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO-B11-15.

In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO-anti-B Lymphocyte Stimulator-6D08-18.

In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO-anti-B Lymphocyte Stimulator-116A01-60.

In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line IO26C04K.

In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line IO50A12.

In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO-B11.

In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide. In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by between 1% and 10% in a competitive inhibition assay. In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by between 1% and 10% in a competitive inhibition assay.

In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 10% and up to 20% in a competitive inhibition assay.

In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 20% and up to 30% in a competitive inhibition assay.

In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 30% and up to 40% in a competitive inhibition assay.

In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 40% and up to 50% in a competitive inhibition assay.

In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 50% and up to 60% in a competitive inhibition assay.

In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 60% and up to 70% in a competitive inhibition assay.

In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 70% and up to 80% in a competitive inhibition assay.

In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 80% and up to 90% in a competitive inhibition assay.

In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 90% and up to 100% in a competitive inhibition assay.

In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCC™ deposit number PTA-3238 to a B Lymphocyte Stimulator polypeptide.

In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCC™ deposit number PTA-3239 to a B Lymphocyte Stimulator polypeptide.

In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCC™ deposit number PTA-3240 to a B Lymphocyte Stimulator polypeptide.

In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCC™ deposit number PTA-3241 to a B Lymphocyte Stimulator polypeptide.

In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCC™ deposit number PTA-3242 to a B Lymphocyte Stimulator polypeptide.

In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCC™ deposit number PTA-3243 to a B Lymphocyte Stimulator polypeptide.

For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. In a specific embodiment, antibodies of the present invention comprise one or more VH and VL domains corresponding to the human scFvs of the invention and framework regions from another immunoglobulin molecule, preferably a human immunoglobulin molecule. In a specific embodiment, antibodies of the present invention comprise one or more CDRs corresponding to the human scFvs of the invention and framework regions from another immunoglobulin molecule, preferably a human immunoglobulin molecule. In other embodiments, an antibody of the present invention comprises one, two, three, four, five, six or more VL CDRs or VH CDRs corresponding to one or more of the human scFvs referred to in Table 1, or fragments or variants thereof, and framework regions (and, optionally CDRs not derived from the scFvs in Table 1) from a human immunoglobulin molecule. In a preferred embodiment, an antibody of the present invention comprises a VH CDR3, VL CDR3, or both, corresponding to the same scFv, or different scFvs referred to in Table 1, or fragments or variants thereof, and framework regions from a human immunoglobulin.

A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a human antibody and a non-human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Chimeric antibodies comprising one or more CDRs from human species and framework regions from a non-human immunoglobulin molecule (e.g., framework regions from a canine or feline immunoglobulin molecule) can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In a preferred embodiment, chimeric antibodies comprise a human CDR3 having an amino acid sequence of any one of the VH CDR3s or VL CDR3s referred to in Table 1, or a variant thereof, and non-human framework regions or human framework regions different from those of the frameworks in the corresponding scFv disclosed in Table 1. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.)

Further, the antibodies of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” B Lymphocyte Stimulator polypeptides using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444 (1993); and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies of the invention which bind to B Lymphocyte Stimulator and competitively inhibit the binding of B Lymphocyte Stimulator to its receptor (as determined by assays well known in the art such as, for example, that disclosed, infra) can be used to generate antiidiotypes that “mimic” a B Lymphocyte Stimulator ligand/receptor-binding domain and, as a consequence, bind to and neutralize B Lymphocyte Stimulator receptors (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP_(—)001183; and/or BAFF-R—GenBank accession number NP_(—)443177). Such neutralizing anti-idiotypes (including molecules comprising, or alternatively consisting of, antibody fragments or variants, such as Fab fragments of such anti-idiotypes) can be used in therapeutic regimens to neutralize B Lymphocyte Stimulator. For example, such anti-idiotypic antibodies can be used to bind B Lymphocyte Stimulator ligands/receptors, and thereby block B Lymphocyte Stimulator mediated biological activity. Alternatively, anti-idiotypes that “mimic” a B Lymphocyte Stimulator binding domain may bind to B Lymphocyte Stimulator receptor(s) and induce B Lymphocyte Stimulator receptor mediated signalling (e.g., activation of nuclear factor of activated T cells (NF-AT), nuclear factor-kappa B (NF-kappa B), and/or AP-1). Such agonistic anti-idiotypes (including agonistic Fab fragments of these anti-idiotypes) can be used in therapeutic regimens to induce or enhance B Lymphocyte Stimulator receptor mediated signalling. For example, such anti-idiotypic antibodies can be used to bind B Lymphocyte Stimulator ligands/receptors, and thereby stimulate B Lymphocyte Stimulator mediated biological activity (e.g., B cell proliferation and/or immunoglobulin production).

Once an antibody molecule of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) has been chemically synthesized or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, or more generally, a protein molecule, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention may be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

Polynucleotides Encoding an Antibody

The invention provides polynucleotides comprising, or alternatively consisting of, a nucleotide sequence encoding an antibody of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). The invention also encompasses polynucleotides that hybridize under high stringency, or alternatively, under intermediate or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides complementary to nucleic acids having a polynucleotide sequence that encodes an antibody of the invention or a fragment or variant thereof.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Since the amino acid sequences of the scFv antibodies and VH domains, VL domains and CDRs thereof, are known (as described in Table 1), nucleotide sequences encoding these antibodies can be determined using methods well known in the art, i.e., the nucleotide codons known to encode the particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody, of the invention. Such a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence of the antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, one or more of the VH and VL domains referred to in Table 1, or fragments or variants thereof, is inserted within framework regions using recombinant DNA techniques known in the art. In a specific embodiment, one, two, three, four, five, six, or more of the CDRs referred to in Table 1, or fragments or variants thereof, is inserted within framework regions using recombinant DNA techniques known in the art. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions, the contents of which are hereby incorporated by reference in its entirety). Preferably, the polynucleotides generated by the combination of the framework regions and CDRs encode an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically binds to B Lymphocyte Stimulator. Preferably, as discussed supra, polynucleotides encoding variants of antibodies or antibody fragments having one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules, or antibody fragments or variants, lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and fall within the ordinary skill of the art.

Recombinant Expression of an Antibody

Recombinant expression of an antibody of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof (e.g., a heavy or light chain of an antibody of the invention or a portion thereof or a single chain antibody of the invention)), requires construction of an expression vector(s) containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule (e.g., a whole antibody, a heavy or light chain of an antibody, or portion thereof (preferably, but not necessarily, containing the heavy or light chain variable domain)), of the invention has been obtained, the vector(s) for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention (e.g., a whole antibody, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody, or a portion thereof, or a heavy or light chain CDR, a single chain Fv, or fragments or variants thereof), operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464, the contents of each of which are hereby incorporated by reference in its entirety) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

The expression vector(s) is(are) transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing polynucleotide(s) encoding an antibody of the invention (e.g., whole antibody, a heavy or light chain thereof, or portion thereof, or a single chain antibody of the invention, or a fragment or variant thereof), operably linked to a heterologous promoter. In preferred embodiments, for the expression of entire antibody molecules, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 1. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) may be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences may be cloned individually into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, NSO, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT2O and T47D, and normal mammary gland cell line such as, for example, CRL7O3O and HsS78Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Ce1122:8 17 (1980)) genes can be employed in tk−, hgprt− or aprt− cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); TIB TECH 11(5):155-2 15 (May, 1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the coding sequence of the antibody, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain is preferably placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2 197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by recombinant expression, it may be purified by any method known in the art for purification of an immunoglobulin molecule, or more generally, for purification of a protein, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibodies of the present invention may be glycosylated or may be non-glycosylated. In addition, antibodies of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

Antibodies of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310:105-111). For example, a peptide corresponding to a fragment of an antibody of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the antibody polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention encompasses antibodies which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, radioisotopic or affinity label to allow for detection and isolation of the antibody.

Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin biotin; examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include a radioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or other radioisotopes such as, for example, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin.

In specific embodiments, antibodies of the invention may be labeled with Europium. For example, antibodies of the invention may be labelled with Europium using the DELFIA Eu-labeling kit (catalog#1244-302, Perkin Elmer Life Sciences, Boston, Mass.) following manufacturer's instructions.

In specific embodiments, antibodies of the invention are attached to macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to antibodies. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators attached to antibodies of the invention is ¹¹¹In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator attached to antibodies of the invention is ⁹⁰Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). In specific embodiments, the macrocyclic chelator is α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid. In other specific embodiments, the DOTA is attached to the antiboddy of the invention via a linker molecule. Examples of linker molecules useful for conjugating DOTA to an antibody are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated by reference in their entirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents that may be conjugated to antibodies, and methods for making and using them, are hereby incorporated by reference in their entireties.

In one embodiment, antibodies of the invention are labeled with biotin. In other related embodiments, biotinylated antibodies of the invention may be used, for example, as an imaging agent or as a means of identifying one or more B Lymphocyte Stimulator receptor(s) or other coreceptor or ligand molecules.

Also provided by the invention are chemically modified derivatives of antibodies of the invention which may provide additional advantages such as increased solubility, stability and in vivo or in vitro circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the antibody. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include, for example, lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins, e.g., antibodies, via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the antibody or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the antibody.

One may specifically desire antibodies chemically modified at the N-terminus of either the heavy chain or the light chain or both. Using polyethylene glycol as an illustration, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective chemical modification at the N-terminus may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular antibody, e.g., a heavy chain or alight chain. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction of antibody with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the antibody. Thus, the invention includes antibody-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to antibodies using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the antibody by a linker can also be produced by reaction of antibodies with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.

The number of polyethylene glycol moieties attached to each antibody of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated antibodies of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per antibody molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

Antibody Characterization

Antibodies of the present invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be characterized in a variety of ways. In particular, antibodies and related molecules of the invention may be assayed for the ability to immunospecifically bind to B Lymphocyte Stimulator or a fragment of B Lymphocyte Stimulator (e.g., to the soluble form or the membrane-bound form of B Lymphocyte Stimulator) using techniques described herein or routinely modifying techniques known in the art. B Lymphocyte Stimulator or B Lymphocyte Stimulator fragments that may be immunospecifically bound by the compositions of the invention include, but are not limited to, human B Lymphocyte Stimulator (SEQ ID NOS:3228 and/or 3229) or B Lymphocyte Stimulator expressed on human monocytes; murine B Lymphocyte Stimulator (SEQ ID NOS:3230 and/or 3231) or B Lymphocyte Stimulator expressed on murine monocytes; rat B Lymphocyte Stimulator (either the soluble forms as given in SEQ ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey B Lymphocyte Stimulator (e.g., the monkey B Lymphocyte Stimulator polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey B Lymphocyte Stimulator, or B Lymphocyte Stimulator expressed on monkey monocytes) or fragments thereof. Preferably compositions of the invention bind human B Lymphocyte Stimulator (SEQ ID NOS:3228 and/or 3229) or fragments thereof. Assays for the ability of the antibodies of the invention to immunospecifically bind B Lymphocyte Stimulator or a fragment of B Lymphocyte Stimulator may be performed in solution (e.g., Houghten, Bio/Techniques 13:412-421 (1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S. Pat. No. 5,223,409), on spores (e.g., U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991)) (each of these references is incorporated herein in its entirety by reference). Antibodies that have been identified to immunospecifically bind to B Lymphocyte Stimulator or a fragment of B Lymphocyte Stimulator can then be assayed for their specificity and affinity for B Lymphocyte Stimulator or a fragment of B Lymphocyte Stimulator using or routinely modifying techniques described herein or otherwise known in the art.

The antibodies of the invention may be assayed for immunospecific binding to B Lymphocyte Stimulator and cross-reactivity with other antigens by any method known in the art. In particular, the ability of an antibody to immunospecifically bind to the soluble form or membrane-bound form of B Lymphocyte Stimulator and the specificity of the antibody, fragment, or variant for B Lymphocyte Stimulator polypeptide from a particular species (e.g., murine, monkey or human, preferably human) may be determined using or routinely modifying techniques described herein or otherwise known in art.

Immunoassays which can be used to analyze immunospecific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40 degrees C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 40 degrees C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96-well microtiter plate with the antigen, washing away antigen that did not bind the wells, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, washing away unbound antibodies or non-specifically bound antibodies, and detecting the presence of the antibodies specifically bound to the antigen coating the well. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, the detectable molecule could be the antigen conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase). One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody (including an scFv or other molecule comprising, or alternatively consisting of, antibody fragments or variants thereof) to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of the present invention for B Lymphocyte Stimulator and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, B Lymphocyte Stimulator is incubated with an antibody of the present invention conjugated to a labeled compound (e.g., ³H or ¹²⁵I) in the presence of increasing amounts of an unlabeled second anti-B Lymphocyte Stimulator antibody.

In a preferred embodiment, BIAcore kinetic analysis is used to determine the binding on and off rates of antibodies (including an scFv or other molecule comprising, or alternatively consisting of, antibody fragments or variants thereof) to B Lymphocyte Stimulator, or fragments of B Lymphocyte Stimulator. BIAcore kinetic analysis comprises analyzing the binding and dissociation of B Lymphocyte Stimulator from chips with immobilized antibodies on their surface as described in detail in Examples 6, 12, 17 and 18, infra.

The antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) can also be assayed for their ability to inhibit, increase, or not significantly alter, the binding of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP_(—)001183; and/or BAFF-R—GenBank accession number NP_(—)443177) using techniques known to those of skill in the art. For example, cells expressing a receptor for B Lymphocyte Stimulator (e.g., IM9, REH, ARH-77 cells, Namalwa, and RPMI-8226 B cell tumor lines as wells as peripheral CD20+ B cells) can be contacted with B Lymphocyte Stimulator in the presence or absence of an antibody, and the ability of the antibody to inhibit, increase, or not significantly alter, B Lymphocyte Stimulator binding to the cells can be measured. B Lymphocyte Stimulator binding to cells can be measured by, for example, flow cytometry or a scintillation assay. B Lymphocyte Stimulator or the antibody can be labeled with a detectable compound such as a radioactive label (e.g., ³²P, ³⁵S, and ¹²⁵I) or a fluorescent label (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) to enable detection of an interaction between B Lymphocyte Stimulator and a B Lymphocyte Stimulator receptor and/or B Lymphocyte Stimulator and an antibody of the invention. Alternatively, the ability of antibodies of the invention to inhibit, increase, or not significantly alter, B Lymphocyte Stimulator binding to a B Lymphocyte Stimulator receptor can be determined in cell-free assays. For example, native or recombinant B Lymphocyte Stimulator (e.g., that having the amino acid sequence of amino acids 134-285 of SEQ ID NO:3228) or a fragment thereof can be contacted with an antibody and the ability of the antibody to inhibit, increase, or not significantly alter, B Lymphocyte Stimulator from binding to a B Lymphocyte Stimulator receptor can be determined Preferably, the antibody is immobilized on a solid support and B Lymphocyte Stimulator or a B Lymphocyte Stimulator fragment is labeled with a detectable compound. Alternatively, B Lymphocyte Stimulator or a B Lymphocyte Stimulator fragment is immobilized on a solid support and the antibody is labeled with a detectable compound. B Lymphocyte Stimulator may be partially or completely purified (e.g., partially or completely free of other polypeptides) or part of a cell lysate. Further, the B Lymphocyte Stimulator polypeptide may be a fusion protein comprising B Lymphocyte Stimulator or a biologically active portion thereof and a domain such as an Immunoglobulin Fc or glutathionine-S-transferase. For example, amino acid residues 1-154 of TACI (GenBank accession number AAC51790), amino acids 1-48 of BCMA (GenBank accession number NP_(—)001183) or amino acids 1-81 of BAFF-R (GenBank Acession Number NP_(—)443177) may be fused to the Fc region of an IgG molecule and used in a cell free assay to determine the ability of antibodies of the invention to inhibit, increase, or not significantly alter, B Lymphocyte Stimulator binding to a B Lymphocyte Stimulator receptor. Alternatively, B Lymphocyte Stimulator can be biotinylated using techniques well known to those of skill in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.).

The antibodies of the invention (including scFvs or other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), can also be assayed for their ability to inhibit, stimulate, or not significantly alter, B Lymphocyte Stimulator-induced B-cell proliferation using techniques known to those of skill in the art. For example, B-cell proliferation can be assayed by ³H-thymidine incorporation assays and trypan blue cell counts (see, e.g., Moore et al., Science 285: 260-263 (1999)). Further, the antibodies of the invention, or fragments or variants thereof, can be assayed for their ability to block, stimulate, or not significantly alter, B Lymphocyte Stimulator-induced activation of cellular signaling molecules and transcription factors such as calcium-modulator and cyclophilin ligand (“CAML”), calcineurin, nuclear factor of activated T cells transcription factor (“NF-AT”), nuclear factor-kappa B (“NF-kappa B”), and AP-1 using techniques known to those of skill in the art (see, e.g., von Bulow and Bram, Science 278:138-141 (1997)). For example, NF-AT activity can be determined by electromobility gel shift assays, by detecting the expression of a protein known to be regulated by NF-AT (e.g., IL-2 expression), by detecting the induction of a reporter gene (e.g., an NF-AT regulatory element operably linked to a nucleic acid encoding a detectable marker such as luciferase, beta-galactosidase or chloramphenicol acetyltransferase (CAT)), or by detecting a cellular response (e.g., cellular differentiation, or cell proliferation).

The antibodies of the invention, or fragments or variants thereof can also be assayed for their ability to neutralize, enhance, or not significantly alter, B Lymphocyte Stimulator activity. For example, antibodies or fragments or variants thereof, may be routinely tested for their ability to inhibit B Lymphocyte Stimulator from binding to cells expressing the receptor for B Lymphocyte Stimulator (see Example 3, infra).

Selection and Screening for Antibodies that Immunospecifically Bind to Soluble B Lymphocyte Stimulator

Antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be screened in a variety of assays to identify those antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator. In one particular assay, antibodies that bind to the biotinylated soluble form of B Lymphocyte Stimulator in solution are captured on streptavidin coated magnetic beads. This assay may be relatively applied to identify antibodies of the invention that neutralize and/or bind to B Lymphocyte Stimulator. Additionally, antibodies may be assayed in neutralization assays described herein or otherwise known in the art (see Example 3, infra). For example, antibodies may be tested for their ability to inhibit soluble B Lymphocyte Stimulator (e.g., biotinylated B Lymphocyte Stimulator) from binding to IM9 cells. In this assay, labeled soluble B Lymphocyte Stimulator (e.g., biotinylated B Lymphocyte Stimulator) is incubated with candidate anti-B Lymphocyte Stimulator antibodies to allow for the formation of B Lymphocyte Stimulator-anti-B Lymphocyte Stimulator antibody complexes. Following incubation, an aliquot of the B Lymphocyte Stimulator-anti-B Lymphocyte Stimulator antibody sample is added to IM9 cells. The binding of soluble B Lymphocyte Stimulator may be determined using techniques known in the art. For example, the binding of biotinylated B Lymphocyte Stimulator to IM9 cells may be detected using a fluorimeter following the addition of streptavidin-delfia. Biotinylated B Lymphocyte Stimulator, if it is not bound by antibodies that neutralize B Lymphocyte Stimulator, binds to the cells is detected. Thus, an antibody that decreases the amount of bio-B Lymphocyte Stimulator that binds to IM-9 cells (relative to a control sample in which the B Lymphocyte Stimulator had been preincubated with an irrelevant antibody or no antibody at all) is identified as one that binds to and neutralizes the soluble form of B Lymphocyte Stimulator. In another assay, antibodies are screened using ELISAs for those antibodies that bind to biotinylated soluble B Lymphocyte Stimulator, but do not bind membrane-bound B Lymphocyte Stimulator, such as, for example, B Lymphocyte Stimulator on membranes from U937 cells (see Examples 2 and 9, infra). In these assays, soluble B Lymphocyte Stimulator (e.g., biotinylated B Lymphocyte Stimulator) and membrane-bound B Lymphocyte Stimulator (e.g., on U937 membranes) are incubated in separate samples with the same antibodies and those antibodies that bind to the soluble B Lymphocyte Stimulator (biotinylated B Lymphocyte Stimulator), but not membrane-bound B Lymphocyte Stimulator (e.g., on U937 membranes) are captured and identified.

Antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be tested to identify those antibodies that do not cross-react with APRIL, endokine-alpha, VEGI, TRAIL, TNF-alpha, TNF-beta, Fas-L, LIGHT, and PBS (see Example 4, infra). Antibodies may also be tested for their affinity for B Lymphocyte Stimulator using, for example, BIAcore analysis (see Examples 6, 12, 17 and 18 infra). Antibodies may also be tested for their ability to stimulate, inhibit, or not alter, B Lymphocyte Stimulator-induced immunoglobulin production and/or B-cell proliferation using techniques known to those of skill in the art. For example, human B-cells, B Lymphocyte Stimulator and antibodies may be incubated together in 96 well plates and ³H-thymidine incorporation may be measured using a scintillation counter.

Selection and Screening for Antibodies that Immunospecifically Bind to Membrane-Bound B Lymphocyte Stimulator

Antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be screened in a variety of assays to identify those antibodies that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator. In one particular assay, antibodies that bind to B Lymphocyte Stimulator on U937 membranes or immobilized histidine-tagged B Lymphocyte Stimulator are captured. Other cell lines that express B Lymphocyte Stimulator that might be useful for testing antibody binding to membrane-bound form of B Lymphocyte Stimulator include, K-562, HL-60 and THP-1 cells. In another assay, antibodies are screened using ELISAs for those antibodies (or antibody fragments or variants) that bind to B Lymphocyte Stimulator on U937 membranes or to histidine-tagged B Lymphocyte Stimulator. In this assay, antibodies are added to 96 well plates coated with U937 membranes or histidine-tagged B Lymphocyte Stimulator and those antibodies or antibody fragments or variants that bind to the U937 membranes or histidine-tagged B Lymphocyte Stimulator are captured. In another assay, antibodies are screened using ELISAs for those antibodies (or antibody fragments or variants thereof) that do not bind to biotinylated B Lymphocyte Stimulator (soluble B Lymphocyte Stimulator) but bind to membrane-bound B Lymphocyte Stimulator, such as, for example, that on membranes from U937 cells (see Example 2, infra). In these assays, soluble B Lymphocyte Stimulator (e.g., biotinylated B Lymphocyte Stimulator) and membrane-bound B Lymphocyte Stimulator (e.g., on U937 membranes) are incubated in separate samples with the same antibodies (or antibody fragments or variants) and those antibodies (or antibody fragments or variants) that do not bind to the soluble B Lymphocyte Stimulator (biotinylated B Lymphocyte Stimulator), but bind the membrane-bound B Lymphocyte Stimulator (e.g., on U937 membranes) are captured and identified. In other assays, antibodies are screened using ELISAs to determine which of the antibodies (or antibody fragments or variants) that bind to histidine-tagged B Lymphocyte Stimulator or membranes from U937 cells do not cross-react with APRIL, endokine-alpha, VEGI, TRAIL, TNF-alpha, TNF-beta, Fas-L, LIGHT, and PBS (See Example 4, infra). ELISAs can also be used to determine which of the antibodies (or antibody fragments or variants) that bind to histidine-tagged B Lymphocyte Stimulator or membranes from U937 cells bind to B Lymphocyte Stimulator in the presence of TNF-alpha (see Example 4, infra). Antibodies or fragments or variants thereof that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator may also be tested for their affinity for histidine-tagged B Lymphocyte Stimulator using high-throughput BIAcore analysis (see Example 14, infra).

Additionally, antibodies of the invention may be screened against cells engineered to express an “uncleavable” form of B Lymphocyte Stimulator in order to determine their specificity for the membrane-bound form of B Lymphocyte Stimulator. Mutations in B Lymphocyte Stimulator which may achieve this result include, but are not limited to, the mutation or deletion of amino acid residues Lys-132 and/or Arg-133 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228. A typical mutagenesis might include mutation of one or both of residues Lys-132 or Arg-133 to alanine residues. Cells expressing such an “uncleavable” form of B Lymphocyte Stimulator provide a profound reagent to use in assaying the ability of antibodies to bind the membrane-bound form of B Lymphocyte Stimulator.

Selection and Screening for Antibodies that Immunospecifically Bind to Soluble and Membrane-Bound B Lymphocyte Stimulator

Antibodies of the invention (including scFvs and other molecules comprising, or alternately consisting of, antibody fragments or variants) may be screened in a variety of assays to identify those antibodies or antibody fragments or variants that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator. In one particular assay, antibodies that bind to immobilized B Lymphocyte Stimulator are captured. In another assay, antibodies are screened using ELISAs for those antibodies (or antibody fragments or variants) that inhibit the binding of soluble B Lymphocyte Stimulator (e.g. soluble bio-B Lymphocyte Stimulator) to IM-9 cells as described supra. In other assays, antibodies are screened using ELISAs for those antibodies that bind to membranes from U937 cells. Additionally, further ELISA assays may be performed using techniques known in the art to determine which antibodies do not cross-react with APRIL, endokine-alpha, VEGI, TRAIL, TNF-alpha, TNF-beta, Fas-L, LIGHT, and PBS, or those antibodies that bind to B Lymphocyte Stimulator in the presence of TNF-alpha (see Example 4 infra). Antibodies may be assayed in neutralization assays using techniques described herein or otherwise known in the art. Antibodies that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator may also be tested for their affinity for B Lymphocyte Stimulator using high-throughput BIAcore analysis.

Antibody Conjugates

The present invention encompasses antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous polypeptide (or portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, antibodies of the invention may be used to target heterologous polypeptides to particular cell types (e.g., cells of monocytic lineage and B-cells), either in vitro or in vivo, by fusing or conjugating the heterologous polypeptides to antibodies of the invention that are specific for particular cell surface antigens (e.g., membrane-bound B Lymphocyte Stimulator on cells of monocytic lineage) or which bind antigens that bind particular cell surface receptors (e.g., TACI, BCMA, BAFF-R located on B cells). Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/2 1232; EP 439,095; Naramura et al., Immunol Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.

In one embodiment, a fusion protein comprises a polypeptide having an amino acid sequence of any one of the VH domains referred to in Table 1, and a heterologous polypeptide. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VH CDR1s referred to in Table 1, and a heterologous polypeptide. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VH CDR2s referred to in Table 1, and a heterologous polypeptide. In a preferred embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VH CDR3s referred to in Table 1 (i.e., SEQ ID NOS:2129-3227), and a heterologous polypeptide.

In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VL domains referred to in Table 1, and a heterologous polypeptide. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VL CDR1s referred to in Table 1, and a heterologous polypeptide. In yet another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VL CDR2s referred to in Table 1, and a heterologous polypeptide. In a preferred embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VL CDR3s referred to in Table 1, and a heterologous polypeptide.

In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1, and one or more VL domains referred to in Table 1, and a heterologous polypeptide. In another embodiment, a fusion protein of the present invention comprises a polypeptide having the amino acid sequence of any one of the VH CDRs referred to in Table 1, and any one of the VL CDRs referred to in Table 1, and a heterologous polypeptide.

The present invention further includes compositions comprising, or alternatively consisting of, heterologous polypeptides fused or conjugated to antibody fragments. For example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, or a portion thereof. Methods for fusing or conjugating polypeptides to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), such methods can be used to generate antibodies with altered activity (e.g., antibodies with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et at., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, polynucleotides encoding antibodies of the invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more portions of a polynucleotide encoding an antibody which portions immunospecifically bind to B Lymphocyte Stimulator may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies of the present invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), can be fused to marker sequences, such as a polypeptides to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine polypeptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Ce1137:767 (1984)) and the “flag” tag (DYKDDDDK, (SEQ ID No: 3238) Stratagene, La Jolla, Calif.).

The present invention further encompasses antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor or prognose the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include, but are not limited to, streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes, but is not limited to, luminol; examples of bioluminescent materials include, but are not limited to, luciferase, luciferin, and aequorin; and examples of suitable radioactive material include, but are not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³ Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶ Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin.

Further, an antibody of the invention (including an scFv or other molecule comprising, or alternatively consisting of, antibody fragments or variants thereof), may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi. In specific embodiments, antibodies of the invention are attached to macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In preferred embodiments, the radiometal ion associated with the macrocyclic chelators attached to antibodies of the invention is ¹¹¹In. In preferred embodiments, the radiometal ion associated with the macrocyclic chelators attached to antibodies of the invention is ⁹⁰Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). In other specific embodiments, the DOTA is attached to the antibody of the invention via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated by reference in their entirety.

A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells and includes such molecules as small molecule toxins and enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof. Examples include, but are not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide (VP-16), tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, thymidine kinase, endonuclease, RNAse, and puromycin and frragments, variants or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine), improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide trimethylolomelamine, chlornaphazine, cholophosphamide, estramustine, ifosfamide, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, chlorozotocin, fotemustine, nimustine, ranimustine, aclacinomysins, azaserine, cactinomycin, calichearnicin, carabicin, caminomycin, carzinophilin, chromomycins, detorubicin, 6-diazo-5-oxo-L-norleucine, epirubicin, esorubicin, idarubicin, marcellomycin, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, quelamycin, rodorubicin, streptonigrin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, thiamiprine, ancitabine, azacitidine, 6-azauridine, carmofur, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptiniurn acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSKO, razoxane, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, urethan, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), taxoids, e.g. paclitaxel (TAXOL″, Bristol-Myers Squibb Oncology, Princeton, N.J.) doxetaxel (TAXOTERE″, Rh6ne-Poulenc Rorer, Antony, France), gemcitabine, ifosfamide, vinorelbine, navelbine, novantrone, teniposide, aminopterin, xeloda, ibandronate, CPT-I 1, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoic acid, esperamicins, capecitabine, and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4 hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, toremifene (Fareston), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Techniques known in the art may be applied to label antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety) and direct coupling reactions (e.g., Bolton-Hunter and Chloramine-T reaction).

The antibodies of the invention which are conjugates can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, but are not limited to, for example, a toxin such as abrin, ricin A, alpha toxin, pseudomonas exotoxin, or diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (see, International Publication No. WO 97/33899), AIM II (see, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (see, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors.

In specific embodiments, antibodies of the invention are conjugated or fused to a polypeptide cytotoxin. An example of a suitable polypeptide cytotoxin is a ribosome-inactivating protein. Type I ribosome-inactivating proteins are single-chain proteins, while type II ribosome-inactivating proteins consist of two nonidentical subunits (A and B chains) joined by a disulfide bond (for a review, see Soria et al., Targeted Diagn. Ther. 7:193 (1992)). In one embodiment, type I ribosome-inactivating proteins that may be used include, but are not limited to, polypeptides from Saponaria officinalis (e.g., saporin-1, saporin-2, saporin-3, saporin-6), Momordica charantia (e.g, momordin), Byronia dioica (e.g., bryodin, bryodin-2), Trichosanthes kirilowii (e.g., trichosanthin, trichokirin), Gelonium multiflorum (e.g., gelonin), Phytolacca americana (e.g., pokeweed antiviral protein, pokeweed antiviral protein-II, pokeweed antiviral protein-S), Phytolacca dodecandra (e.g., dodecandrin, Mirabilis antiviral protein). Ribosome-inactivating proteins are described, for example, by Walsh et al., U.S. Pat. No. 5,635,384. In specific embodiments, an antibody of the invention is conjugated or fused to a fragment or variant of the ribosome inactivating proteins described above, particularly when said fragment or variant retains activity.

In another embodiment, type II ribosome-inactivating proteins that may be used according to the invention include, but are not limited to, polypeptides from Ricinus communis (e.g., ricin), Abrus precatorius (e.g., abrin), Adenia digitata (e.g., modeccin). Since type II ribosome-inactiving proteins include a B chain that binds galactosides and a toxic A chain that depurinates adensoine. Type II ribosome-inactivating protein conjugates should include the A chain. Additional ribosome-inactivating proteins that may be used according to the invention include, but are not limited to, bouganin, clavin, maize ribosome-inactivating proteins, Vaccaria pyramidata ribosome-inactivating proteins, nigrine b, basic nigrine 1, ebuline, racemosine b, luffin-a, luffin-b, luffin-S, and other ribosome-inactivating proteins known to those of skill in the art. See, for example, Bolognesi and Stirpe, International Publication No. WO98/55623, Colnaghi et al., International Publication No. WO97/49726, Hey et al., U.S. Pat. No. 5,635,384, Bolognesi and Stirpe, International Publication No. WO95/07297, Arias et al., International Publication No. WO94/20540, Watanabe et al., J. Biochem. 106:6 977 (1989); Islam et al., Agric. Biol. Chem. 55:229 (1991), and Gao et al., FEBS Lett. 347:257 (1994). In specific embodiments, an antibody of the invention is conjugated or fused to a fragment or variant of the ribosome inactivating proteins described above, particularly when said fragment or variant retains activity.

Additional ribosome inactivating proteins (RIPs) that may be conjugated or fused to antibodies of the invention include, but are not limited to, Type I Plant RIPs such as Pokeweed antiviral proteins, Tritin, Gelonin, Momordin, Saporin, Dianthin, and Maize RIP; Type II Plant RIPs such as Ricin, Abrin, Modecin, Viscumin, Volkensin, Cinnamomin, Mistletoe lectin I and Luffangulin (6 kda); Bacterial RIPS such as Shiga toxin, and Shiga-like toxin; and Fungal RIPS such as alpha-sarcin, mitogillin, and restrictocin. In specific embodiments, an antibody of the invention is conjugated or fused to a fragment or variant of the ribosome inactivating proteins described above, particularly when said fragment or variant retains activity.

Ricin A homologues that may be conjugated or fused to antibodies of the invention include, but are not limited to, polypeptides that have the same amino acid sequence as a protein selected from the group consisting of: type 2 ribosome-inactivating protein cinnamomin III precursor, abrin-d precursor from Indian licorice or fragment thereof, RIP precursor of Sambucus nigra, RIP bryodin II precursor, alpha-trichosanthin, ribosome-inactivating protein precursor from Sambucus ebulus, karasurin-B, Trichobakin, Beta-Luffin, Beta-galactoside specific lectin I A chain (MLA; ML-I A), lectin chain A isoform 2 from Viscum album subsp. coloratum, curcin precursor from Jatropha curcas, trichoanguina from snake gourd, ribosome-inactivating protein IRAb from Iris hollandica, Momordin, trichoanguin from Trichosanthes cucumerina, momordin II from Momordica balsamina, gelonin from Gelonium multiflorum, ribosome inactivating protein RIPm from Polygonatum multiflorum, ribosome inactivating protein Euserratin 2 precursor from Euphorbia serrata, alpha-PAP (pokeweed antiviral protein) from Phytolacca americana, ribosome-inactivating protein gynostemmin II from Gynostemma pentaphyllum, trichosanthin, ribosome-inactivating protein 2 from Phytolacca insularis, bouganin from Bougainvillea spectabilis, antiviral protein from Clerodendrum aculeatum, anti-viral protein PAP from Phytolacca acinosa, type I RIP moschatin I, antiviral ribosome inactivating protein from Chenopodium album, Betavulgin from Beta vulgaris subsp. vulgaris, ribosome-inactivating protein benincasin from Benincasa hispida, ribosome-inactivating protein ME1 from Mirabilis expansa, MAP-MIRABILIS—Four-o'clock (Marvel-of-peru), RIP amaranthin, amarandin-S from Amaranthus tricolor, ribosome-inactivating protein from Spinacia oleracea, type 1 RIP musarmin 3 from Muscari, saporin from Saponaria officinalis, dianthin 30 from Dianthus caryophyllus, and tritin from Triticum aestivum, or biologically active fragments or variants thereof.

Other toxins that may be conjugated or fused to antibodies of the invention include, but are not limited to diptheria toxin, Pseudomonas exotoxin A, Botulinum neurotoxin, Clostridium perfringens epsilon toxin, Conotoxins, and Staphylococcal enterotoxins.

Antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating a therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody of the invention can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody of the invention (including an scFv or and other molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

Certain forms of antibodies, particularly, IgG3 and IgM antibodies, as well as IgG1 and IgG2 antibodies to a lesser extent, are able to mediate cellular lysis via the complement cascade. Thus antibodies of the invention may be used to kill cells expressing Neutrokine-alpha on their surface or cells bearing Neutrokine-alpha receptors via complement-mediated killing.

Antibodies of the invention may be used to mediate antibody dependent cell-mediated cytotocicity (ADCC) by, for example, Natural Killer (NK) cells. ADCC occurs when antibodies of the IgG1 or IgG3 isotype interact with FcγRIII receptor on Natural Killer cells triggering an cytotoxic attack by the NK cell on the antibody coated cells. Thus antibodies of the invention may be used to kill cells expressing Neutrokine-alpha on their surface or cells bearing Neutrokine-alpha receptors via ADCC.

Use of Antibodies for Epitope Mapping

The present invention provides antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that can be used to identify epitopes of B Lymphocyte Stimulator. In particular, the antibodies of the present invention can be used to identify epitopes of human B Lymphocyte Stimulator (SEQ ID NOS:3228 and/or 3229) or B Lymphocyte Stimulator expressed on human monocytes; murine B Lymphocyte Stimulator (SEQ ID NOS:3230 and/or 3231) or B Lymphocyte Stimulator expressed on murine monocytes; rat B Lymphocyte Stimulator (either the soluble forms as given in SEQ ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey B Lymphocyte Stimulator (e.g., the monkey B Lymphocyte Stimulator polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey B Lymphocyte Stimulator, or B Lymphocyte Stimulator expressed on monkey monocytes) using techniques described herein or otherwise known in the art. Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211.)

Diagnostic Uses of Antibodies

Labeled antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample from an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase or decrease in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of aberrant expression.

By “biological sample” is intended any fluids and/or cells obtained from an individual, body fluid, body tissue, body cell, cell line, tissue culture, or other source which may contain B Lymphocyte Stimulator protein or mRNA. Body fluids include, but are not limited to, sera, plasma, urine, synovial fluid, spinal fluid, saliva, and mucous. Tissues samples may be taken from virtually any tissue in the body. Tissue samples may also be obtained from autopsy material. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

The invention also provides for the detection of aberrant expression of B Lymphocyte Stimulator receptor comprising (a) assaying the expression of B Lymphocyte Stimulator receptor in a biological sample from an individual using one or more antibodies or fragments or variants thereof that immunospecifically binds only to soluble B Lymphocyte Stimulator, but does not inhibit B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor binding. Such an antibody, by way of an example that is not to be construed as limiting, would be one that is able to capture a biotinylated B Lymphocyte Stimulator from solution (see Example 8), but that would not prevent B Lymphocyte Stimulator from binding to IM-9 cells (see Example 3). and (b) comparing the level of B Lymphocyte Stimulator receptor with a standard level of B Lymphocyte Stimulator receptor, e.g., in normal tissue or cell samples, whereby an increase or decrease in the assayed level of B Lymphocyte Stimulator receptor compared to the standard level of B Lymphocyte Stimulator receptor is indicative of aberrant expression.

Antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor autoimmune disorders and/or immunodeficiencies, and/or diseases or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample from an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase or decrease in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of an autoimmune disorder or disease and/or an immunodeficiency. In specific embodiments, an increase in the assayed level of B Lymphocyte Stimulator is indicative of an autoimmune disorder or disease. In other specific embodiments, a decrease in the assayed level of B Lymphocyte Stimulator is indicative of an immunodeficiency.

Antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to B Lymphocyte Stimulator but, do not inhibit B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor binding can be used for diagnostic purposes to detect, diagnose, prognose, or monitor autoimmune disorders and/or immunodeficiencies, and/or diseases or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator receptor comprising: (a) assaying the expression of B Lymphocyte Stimulator receptor in a biological sample from an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator receptor with a standard level of B Lymphocyte Stimulator receptor, e.g., in normal biological samples, whereby an increase or decrease in the assayed level of B Lymphocyte Stimulator receptor compared to the standard level of B Lymphocyte Stimulator receptor is indicative of an autoimmune disorder or disease and/or an immunodeficiency. In specific embodiments, an increase in the assayed level of B Lymphocyte Stimulator receptor is indicative of an autoimmune disorder or disease. In other specific embodiments, a decrease in the assayed level of B Lymphocyte Stimulator receptor is indicative of an immunodeficiency.

Autoimmune disorders, diseases, or conditions that may be detected, diagnosed, prognosed, or monitored using the antibodies of the invention include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, dilated cardiomyopathy, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, diabetes mellitus (e.g. Type I diabetes mellitus or insulin dependent diabetes mellitis), juvenile onset diabetes, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia (Addison's disease), idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjögren's syndrome, diabetes millitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulomatous, degenerative, and atrophic disorders and other disorders such as inflammatory skin diseases including psoriasis and sclerosis, responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), respiratory distress syndrome (including adult respiratory distress syndrome, ARDS), meningitis, encephalitis, colitis, allergic conditions such as eczema and other conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, leukocyte adhesion deficiency, Reynaud's syndrome, and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, granulomatosis and diseases involving leukocyte diapedesis, central nervous system (CNS) inflammatory disorder, multiple organ injury syndrome, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, Lambert-Eaton myasthenic syndrome, Beheet disease, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies or autoimmune thrombocytopenia etc.

In specific embodiments, the present invention encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some immunodeficiencies). In other specific embodiments, the present invention encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency).

Immunodeficiencies that may be detected, diagnosed, prognosed, or monitored using the antibodies of the invention include, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency.

Elevated levels of soluble B Lymphocyte Stimulator have been observed in the serum of patients with Systemic Lupus Erythematosus (SLE). In comparing the sera of 150 SLE patients with that of 38 control individuals, it was found that most of the SLE patients had more than 5 ng/ml of serum B Lymphocyte Stimulator, more than 30% of SLE patients had levels greater than 10 ng/ml, and approximately 10% of SLE patients had serum B Lymphocyte Stimulator levels greater than 20 ng/ml. In contrast, the majority of normal controls had B Lymphocyte Stimulator levels less than 5 ng/ml, and less than 10% had levels higher than 10 ng/ml. The elevated levels of B Lymphocyte Stimulator protein in sera is present in the soluble form and has biologic activity as assayed by the ability to stimulate anti-IgM treated B cells in vitro. SLE patients with more than 15 ng/ml serum B Lymphocyte Stimulator were also found to have elevated levels of anti-dsDNA antibodies compared to both normal controls and SLE patients with less than 5 ng/ml of serum B Lymphocyte Stimulator. (unpublished data).

In addition the serum of two subgroups of patients which were positive for anti-nuclear antibodies (ANA+) but did not meet the formal requirements of the American College of Rheumatology (ACR) for classification of SLE were analyzed for B Lymphocyte Stimulator levels. The first subgroup of sera was ANA+ sera that came from patients who did not present with the clinical impression of SLE. This group had only slightly elevated levels of B Lymphocyte Stimulator (˜9 ng/ml B Lymphocyte Stimulator). The second subgroup however, which was ANA+ sera from patients who presented with the clinical impression of SLE, had significantly increased B Lymphocyte Stimulator levels (˜15 ng/ml). These results suggest that an elevated level of B Lymphocyte Stimulator precedes the formal fulfillment of the ACR criteria. The ACR criteria are described in Tan, E. M., et al, Arthritis and Rheumatism 25:1271-1277 (1982).

Thus in specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Systemic Lupus Erythematosus or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of SLE.

In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor IgA nephropathy or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of IgA nephropathy.

In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Sjögren's Syndrome or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of Sjögren's Syndrome.

In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor HIV infection or conditions associated therewith (e.g. AIDS). The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of HIV infection.

In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Myasthenia Gravis or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of Myasthenia Gravis.

In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor idiopathic thrombocytopenic purpura (ITP) or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of idiopathic thrombocytopenic purpura (ITP).

In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor hemolytic anemia or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of hemolytic anemia.

In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor thyroiditis or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of thyroiditis.

In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Goodpasture's syndrome or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of Goodpasture's syndrome.

In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor multiple sclerosis or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of multiple sclerosis.

In additional embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Rheumatoid Arthritis. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample (e.g., serum and synovial fluid) of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of Rheumatoid arthritis.

In additional embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor an immune-based rheumatologic disease, (e.g., SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by calcinosis, Raynaud's phenomenon, esophageal motility disorders, sclerodactyl), and telangiectasia), Seronegative spondyloarthropathy (SpA), Polymyositis/dermatomyositis, Microscopic polyangiitis, Hepatitis C-associated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder). The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample (e.g., serum and synovial fluid) of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of monitor an immune-based rheumatologic disease.

It has been observed, that serum B Lymphocyte Stimulator levels inversely correlate with nephrotic range proteinuria (>3 gm proteinuria in a 24 hour urine collection) using a sample of 71 SLE patients (p=0.019). Proteinuria was determined in 71 SLE patients within one month of phlebotomy for serum B Lymphocyte Stimulator determination. Serum B Lymphocyte Stimulator was classified as low, normal, or high based on the 5^(th) through 95^(th) percentiles for normal controls. Nephrotic-range proteinuria was inversely correlated with serum Neutrokine-alpha levels. Thus, in specific embodiments, serum levels of B Lymphocyte Stimulator (determined using one or more antibodies of the present invention) in individuals diagnosed with an immune based rheumatologic disease (e.g., SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by calcinosis, Raynaud's phenomenon, esophageal motility disorders, sclerodactyl), and telangiectasia), seronegative spondyloarthropathy (SpA), polymyositis/dermatomyositis, microscopic polyangiitis, hepatitis C-asociated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder) may be used to determine, diagnose, prognose, or monitor the severity of certain aspects or symptoms of the disease, such as nephrotic-range proteinuria.

In another specific embodiment, antibodies of the invention are used to diagnose, prognose, treat, or prevent conditions associated with CVID, including, but not limited to, conditions associated with acute and recurring infections (e.g., pneumonia, bronchitis, sinusitis, otitis media, sepsis, meningitis, septic arthritis, and osteomyelitis), chronic lung disease, autoimmunity, granulomatous disease, lymphoma, cancers (e.g., cancers of the breast, stomach, colon, mouth, prostate, lung, vagina, ovary, skin, and melanin forming cells (i.e. melanoma), inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, and ulcerative proctitis), malabsorption, Hodgkin's disease, and Waldenstrom's macroglobulinemia.

The invention provides a diagnostic assay for diagnosing or prognosing a disease or disorder, comprising: (a) assaying for the level of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically bind to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard B Lymphocyte Stimulator level, e.g., in a biological sample from a patient without the disease or disorder, whereby an increase or decrease in the assayed B Lymphocyte Stimulator level compared to the standard level of B Lymphocyte Stimulator is indicative of a particular disease or disorder. With respect to cancer, the presence of a relatively high amount of B Lymphocyte Stimulator in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

In specific embodiments, the presence of a relatively high amount of membrane-bound B Lymphocyte Stimulator in a biological sample is indicative of monocytic cell related leukemias or lymphomas, such as, for example acute myelogenous leukemia and/or the severity thereof.

In other specific embodiments, the presence of a relatively high amount of B Lymphocyte Stimulator receptor in a biological sample (as determined using antibodies of the invention that bind to soluble B Lymphocyte Stimulator, but do not inhibit B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor binding) is indicative of B cell related leukemias or lymphomas (e.g., chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's disease), and/or the severity thereof.

In specific embodiments, the invention provides a diagnostic assay for diagnosing or prognosing Systemic Lupus Erythematosus, comprising: (a) assaying for the level of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically bind to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard B Lymphocyte Stimulator level, e.g., in a biological sample from a patient without Systemic Lupus Erythematosus, whereby an increase in the assayed B Lymphocyte Stimulator level compared to the standard level of B Lymphocyte Stimulator is indicative of Systemic Lupus Erythematosus.

In specific embodiments, the invention provides a diagnostic assay for diagnosing or prognosing a Rheumatoid Arthritis, comprising: (a) assaying for the level of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically bind to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard B Lymphocyte Stimulator level, e.g., in a biological sample from a patient without Rheumatoid Arthritis, whereby an increase or decrease in the assayed B Lymphocyte Stimulator level compared to the standard level of B Lymphocyte Stimulator is indicative of Rheumatoid Arthritis.

The invention provides a diagnostic assay for diagnosing or prognosing a disease or disorder, comprising: (a) assaying for the level of B Lymphocyte Stimulator receptor in cells or a tissue sample of an individual using one or more antibodies of the invention that immunospecifically binds only to soluble B Lymphocyte Stimulator, but does not neutralize B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor binding; and (b) comparing the level of B Lymphocyte Stimulator receptor with a standard B Lymphocyte Stimulator receptor level, e.g., in a tissue sample from a patient without the disease or disorder, whereby an increase or decrease in the assayed B Lymphocyte Stimulator receptor level compared to the standard level of B Lymphocyte Stimulator receptor is indicative of a particular disease or disorder. With respect to cancer, the presence of a relatively high amount of B Lymphocyte Stimulator receptor in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) can be used to assay protein levels in a biological sample using classical immunohistological methods as described herein or as known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, alkaline phosphatase, and horseradish peroxidase; radioisotopes, such as iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically binds to B Lymphocyte Stimulator; b) waiting for a time interval following the administering for permitting the labeled antibody to preferentially concentrate at sites in the subject where B Lymphocyte Stimulator is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled antibody in the subject, such that detection of labeled antibody or fragment thereof above the background level and above or below the level observed in a person without the disease or disorder indicates that the subject has a particular disease or disorder associated with aberrant expression of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ⁹⁹Tc. The labeled antibody will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disorder, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

Immunophenotyping

The antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be utilized for immunophenotyping of cell lines and biological samples by their B Lymphocyte Stimulator expression or B Lymphocyte Stimulator receptor expression. Various techniques can be utilized using antibodies, fragments, or variants of the invention to screen for cellular populations (i.e., immune cells, particularly monocytic cells or B-cells) expressing B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor, and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (see, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e., minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.

In one embodiment, antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) are used to identify cells of monocytic or B cell origin.

Use of Antibodies to Identify, Purify and/or Target B Lymphocyte Stimulator Protein and/or B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor Expressing Cells

The antibodies of the invention may be used to identify, purify and target B Lymphocyte Stimulator protein and/or B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expressing cells. For example, the antibodies of the invention when coupled to a detectable label can be used to identify cells that express B Lymphocyte Stimulator. More specifically, the antibodies of the invention can be used in immunohistochemistry or in flow cytometry. Additionally, the antibodies of the invention may be used to purified B Lymphocyte Stimulator proteins or portions thereof from cells expressing B Lymphocyte Stimulator. In more specific embodiments, antibodies of the invention may be conjugated (directly or indirectly) to a detectable label such a fluorescent label and used in fluorescence activated cell sorting (FACS). Alternatively, antibodies of the invention may be conjugated (directly or indirectly) to a magnetic particle or bead and used in magnetic cell sorting (MACS).

Antibodies of the invention may be used to target therapeutic or toxic moieties to B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells (see Antibody Conjugates section above). In a specific embodiment, antibodies of the invention may also be used to kill B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells. In one embodiment, antibodies of the invention are linked to a cytotoxic moiety which kills B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells that have been contacted with such an antibody conjugate.

Certain forms of antibodies, particularly, IgG3 and IgM antibodies, as well as IgG1 and IgG2 antibodies to a lesser extent, are able to mediate cellular lysis via the complement cascade. Thus antibodies of the invention may be used to kill B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells via complement-mediated killing.

Antibodies of the invention may be used to mediate antibody dependent cell-mediated cytotocicity (ADCC) by, for example, Natural Killer (NK) cells. ADCC occurs when antibodies of the IgG1 or IgG3 isotype interact with FcγRIII receptor on Natural Killer cells triggering an cytotoxic attack by the NK cell on the antibody coated cells. Thus antibodies of the invention may be used to kill B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells via ADCC.

Therapeutic Uses of Antibodies

The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention and nucleic acids encoding antibodies (and anti-idiotypic antibodies) of the invention as described herein. The antibodies of the invention can be used to treat, ameliorate or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant B Lymphocyte Stimulator expression and/or activity or aberrant B Lymphocyte Stimulator receptor expression and/or activity includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

Antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that function as agonists or antagonists of B Lymphocyte Stimulator, preferably of B Lymphocyte Stimulator-induced signal transduction, can be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, lack of B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or lack of B Lymphocyte Stimulator receptor function. For example, antibodies of the invention which disrupt the interaction between B Lymphocyte Stimulator and its receptor may be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive of B Lymphocyte Stimulator receptor function. Antibodies of the invention which do not prevent B Lymphocyte Stimulator from binding its receptor but inhibit or downregulate B Lymphocyte Stimulator-induced signal transduction can be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive B Lymphocyte Stimulator receptor function. In particular, antibodies of the present invention which prevent B Lymphocyte Stimulator-induced signal transduction by specifically recognizing the unbound B Lymphocyte Stimulator, receptor-bound B Lymphocyte Stimulator or both unbound and receptor-bound B Lymphocyte Stimulator can be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive B Lymphocyte Stimulator receptor function. The ability of an antibody of the invention to inhibit or downregulate B Lymphocyte Stimulator-induced signal transduction may be determined by techniques described herein or otherwise known in the art. For example, B Lymphocyte Stimulator-induced receptor activation and the activation of signaling molecules can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or a signaling molecule by immunoprecipitation followed by western blot analysis (for example, as described herein).

In a specific embodiment, an antibody of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that inhibits or downregulates B Lymphocyte Stimulator activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to B Lymphocyte Stimulator activity in absence of the antibody is administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive B Lymphocyte Stimulator receptor function. In another embodiment, a combination of antibodies, a combination of antibody fragments, a combination of antibody variants, or a combination of antibodies, antibody fragments, and/or variants that inhibit or downregulate B Lymphocyte Stimulator activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to B Lymphocyte Stimulator activity in absence of said antibodies, antibody fragments, and/or antibody variants are administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive B Lymphocyte Stimulator receptor function.

Further, antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which activate B Lymphocyte Stimulator-induced signal transduction can be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, lack of B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or lack of B Lymphocyte Stimulator receptor function. These antibodies may potentiate or activate either all or a subset of the biological activities of B Lymphocyte Stimulator-mediated receptor activation, for example, by inducing multimerization of B Lymphocyte Stimulator and/or multimerization of the receptor. The antibodies of the invention may be administered with or without being pre-complexed with B Lymphocyte Stimulator. In a specific embodiment, an antibody of the present invention that increases B Lymphocyte Stimulator activity by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% relative to B Lymphocyte Stimulator activity in absence of the antibody is administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, lack of B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or lack of B Lymphocyte Stimulator receptor function. In another embodiment, a combination of antibodies, a combination of antibody fragments, a combination of antibody variants, or a combination of antibodies, antibody fragments and/or antibody variants that increase B Lymphocyte Stimulator activity by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% relative to B Lymphocyte Stimulator activity in absence of the said antibodies or antibody fragments and/or antibody variants is administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or lack of B Lymphocyte Stimulator function or aberrant B Lymphocyte Stimulator receptor expression or lack of B Lymphocyte Stimulator receptor function.

One or more antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator may be used locally or systemically in the body as a therapeutic. The antibodies of this invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may also be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents, anti-angiogenesis and anti-inflammatory agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments, or variants, (e.g., derivatives), or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator, or polynucleotides encoding antibodies that immunospecifically bind to B Lymphocyte Stimulator, for both immunoassays directed to and therapy of disorders related to B Lymphocyte Stimulator polynucleotides or polypeptides, including fragments thereof. Such antibodies will preferably have an affinity for B Lymphocyte Stimulator and/or B Lymphocyte Stimulator fragments. Preferred binding affinities include those with a dissociation constant or K_(D) less than or equal to 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, or 10⁻⁵ M. More preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with a dissociation constant or K_(D) less than or equal to 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, or 10⁻⁸ M. Even more preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with a dissociation constant or K_(D) less than or equal to 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×−13 M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. The invention encompasses antibodies that bind B Lymphocyte Stimulator polypeptides with a dissociation constant or K_(D) that is within any one of the ranges that are between each of the individual recited values.

In a preferred embodiment, antibodies of the invention neutralize B Lymphocyte Stimulator activity. In another preferred embodiment, antibodies of the invention inhibit B cell proliferation.

In a preferred embodiment, antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) inhibit or reduce binding of the soluble form of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor. In another preferred embodiment antibodies of the invention inhibit or reduce B cell proliferation induced by the soluble form of B Lymphocyte Stimulator. In another preferred embodiment antibodies of the invention inhibit or reduce immunoglobulin production induced by the soluble form of B Lymphocyte Stimulator.

In a preferred embodiment, antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) inhibit or reduce binding of membrane-bound B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor. In another preferred embodiment, antibodies of the invention inhibit or reduce B cell proliferation induced by the membrane-bound form of B Lymphocyte Stimulator. In another preferred embodiment, antibodies of the invention inhibit or reduce immunoglobulin production induced by the membrane bound form of B Lymphocyte Stimulator.

In a preferred embodiment, antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) inhibit or reduce binding of both the soluble and membrane-bound forms of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor. In another preferred embodiment, antibodies of the invention inhibit or reduce B cell proliferation induced by either or both forms of B Lymphocyte Stimulator. In another preferred embodiment, antibodies of the invention inhibit or reduce immunoglobulin production induced by either or both forms of B Lymphocyte Stimulator.

In one embodiment, the invention provides a method of delivering antibody conjugates of the invention to targeted cells, such as, for example, monocytic cells expressing the membrane-bound form of B Lymphocyte Stimulator, or B cells expressing a B Lymphocyte Stimulator receptor.

In one embodiment, the invention provides a method for the specific delivery of antibodies and antibody conjugates of the invention to cells by administering molecules of the invention that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering antibodies or antibody conjugates of the invention (e.g., antibodies conjugated with radioisotopes, toxins, or cytotoxic prodrugs). In a specific embodiment, the invention provides a method for the specific destruction of cells of monocytic lineage (e.g., monocytic cell related leukemias or lymphomas, such as, for example acute myelogenous leukemia) by administering antibodies or antibody conjugates of the invention (e.g., antibodies conjugated with radioisotopes, toxins, or cytotoxic prodrugs) that immunospecifically bind the membrane-bound form of B Lymphocyte Stimulator. In another specific embodiment, the invention provides a method for the specific destruction of cells of B cell lineage (e.g., B cell related leukemias or lymphomas (e.g., chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's disease) by administering antibodies or antibody conjugates of the invention (e.g., antibodies conjugated with radioisotopes, toxins, or cytotoxic prodrugs) that bind soluble B Lymphocyte Stimulator, but do not inhibit B Lymphocyte Stimulator binding to a B Lymphocyte Stimulator receptor on B cells.

In another preferred embodiment antibodies of the invention (including antibody fragments and variants) promote or enhance B cell proliferation induced by the soluble form of B Lymphocyte Stimulator. In another preferred embodiment, antibodies of the invention (including antibody fragments and variants) promote or enhance B cell proliferation induced by the membrane or soluble form of APRIL. In another preferred embodiment antibodies of the invention (including antibody fragments and variants) increase or enhance immunoglobulin production induced by the soluble form of B Lymphocyte Stimulator. In another preferred embodiment antibodies of the invention (including antibody fragments and variants) increase or enhance immunoglobulin production induced by the membrane bound or soluble form of APRIL. In another preferred embodiment antibodies of the invention (including antibody fragments and variants) increase or enhance immunoglobulin production in response to T cell dependent immunogens. In another preferred embodiment antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance immunoglobulin production in response to T cell independent immunogens.

In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate immune disorders Immune disorders include, but are not limited to, autoimmune disorders (e.g., arthritis, graft rejection, Hashimoto's thyroiditis, insulin-dependent diabetes, lupus, idiopathic thrombocytopenic purpura, systemic lupus erythrematosus and multiple sclerosis), elective IgA deficiency, ataxia-telangiectasia, common variable immunodeficiency (CVID), X-linked agammaglobulinemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, idiopathic hyper-eosinophilic syndrome, monocytic leukemoid reaction, monocytic leukocytosis, monocytic leukopenia, monocytopenia, monocytosis, and graft or transplant rejection.

As discussed herein, antibodies and antibody compositions of the invention, may be used to treat, prevent, ameliorate, diagnose or prognose various immune system-related disorders and/or conditions associated with these disorders, in mammals, preferably humans. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of antibody and antibody compositions of the invention that can inhibit an immune response, particularly the proliferation of B cells and/or the production of immunoglobulins, may be an effective therapy in treating and/or preventing autoimmune disorders. Thus, in preferred embodiments, antibodies and antibody compositions of the invention are used to treat, prevent, ameliorate, diagnose and/or prognose an autoimmune disorder, or condition(s) associated with such disorder.

Autoimmune disorders and conditions associated with these disorders that may be treated, prevented, ameliorated, diagnosed and/or prognosed with the therapeutic and pharmaceutical compositions of the invention include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

Additional autoimmune disorders and conditions associated with these disorders that may be treated, prevented, ameliorated, diagnosed and/or prognosed with the therapeutic and pharmaceutical compositions of the invention include, but are not limited to, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis) (often characterized, e.g., by cell-mediated and humoral thyroid cytotoxicity), systemic lupus erhythematosus (often characterized, e.g., by circulating and locally generated immune complexes), discoid lupus, Goodpasture's syndrome (often characterized, e.g., by anti-basement membrane antibodies), Pemphigus (often characterized, e.g., by epidermal acantholytic antibodies), Receptor autoimmunities such as, for example, (a) Graves' Disease (often characterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis (often characterized, e.g., by acetylcholine receptor antibodies), and (c) insulin resistance (often characterized, e.g., by insulin receptor antibodies), autoimmune hemolytic anemia (often characterized, e.g., by phagocytosis of antibody-sensitized RBCs), autoimmune thrombocytopenic purpura (often characterized, e.g., by phagocytosis of antibody-sensitized platelets.

Additional autoimmune disorders and conditions associated with these disorders that may be treated, prevented, ameliorated, diagnosed and/or prognosed with the therapeutic and pharmaceutical compositions of the invention include, but are not limited to, rheumatoid arthritis (often characterized, e.g., by immune complexes in joints), schleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis/dermatomyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes) such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjögren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes millitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies), chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitchondrial antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), inflammatory myopathies, and many other inflammatory, granulomatous, degenerative, and atrophic disorders.

In specific embodiments, antagonistic anti-neutrokine-alpha antibodies are useful in the diagnosis and treatment or prevention of dilated cardiomyopathy.

In a preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, a member of the group: autoimmune hemolytic anemia, as primary glomerulonephritis, IgA glomerulonephritis, Goodpasture's syndrome, idiopathic thrombocytopenia, Multiple Sclerosis, Myasthenia Gravis, Pemphigus, polymyositis/dermatomyositis, relapsing polychondritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, Uveitis, vasculitis, and primary biliary cirrhosis.

In specific embodiments, pharmaceutical compositions of the invention are useful in the diagnosis and treatment or prevention of liver diseases, including but not limited to, primary biliary cirrhosis, primary sclerosing cholangitis, chronic hepatitis C infection, autoimmune hepatitis and alcoholic liver disease. In specific embodiments, antagonists of Neutrokine-alpha, such as an anti-Neutrokine-alpha specific antibody, are useful in the diagnosis and treatment or prevention of liver diseases, including but not limited to, primary biliary cirrhosis, primary sclerosing cholangitis, chronic hepatitis C infection, and alcoholic liver disease.

In another preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, an immune based-rheumatologic disease, such as, for example, SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by calcinosis, Raynaud's phenomenon, esophageal motility disorders, sclerodactyl), and telangiectasia), Seronegative spondyloarthropathy (SpA), polymyositis/dermatomyositis, microscopic polyangiitis, hepatitis C-associated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder.

In a specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, rheumatoid arthritis and/or medical conditions associated therewith.

For example, an antibody, or antibodies, of the present invention are used to treat patients with clinical diagnosis of rheumatoid arthritis (RA). The patient treated preferably will not have a B cell malignancy. Moreover, the patient is optionally further treated with any one or more agents employed for treating RA such as salicylate; nonsteroidal anti-inflammatory drugs such as indomethacin, phenylbutazone, phenylacetic acid derivatives (e.g. ibuprofen and fenoprofen), naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac), halogenated anthranilic acid (meclofenamate sodium), piroxicam, zomepirac and diflunisal; antimalarials such as chloroquine; gold salts; penicillamine; or immunosuppressive agents such as methotrexate or corticosteroids in dosages known for such drugs or reduced dosages. Preferably however, the patient is only treated with an antibody, or antibodies, of the present invention. Antibodies of the present invention are administered to the RA patient according to a dosing schedule as described infra, which may be readily determined by one of ordinary skill in the art. The primary response is determined by the Paulus index (Paulus et al. Athritis Rheum. 33:477-484 (1990)), i.e. improvement in morning stiffness, number of painful and inflamed joints, erythrocyte sedimentation (ESR), and at least a 2-point improvement on a 5-point scale of disease severity assessed by patient and by physician. Administration of an antibody, or antibodies, of the present invention will alleviate one or more of the symptoms of RA in the patient treated as described above.

In a specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, amelioate, diagnose or prognose, lupus and/or medical conditions associated therewith. Lupus-associated conditions that may be treated, prevented, ameliorated, prognosed and/or diagnosed with the antibodies and antibody compositions of the invention include, but are not limited to, hematologic disorders (e.g., hemolytic anemia, leukopenia, lymphopenia, and thrombocytopenia), immunologic disorders (e.g., anti-DNA antibodies, and anti-Sm antibodies), rashes, photosensitivity, oral ulcers, arthritis, fever, fatigue, weight loss, serositis (e.g., pleuritus (pleurisy)), renal disorders (e.g., nephritis), neurological disorders (e.g., seizures, peripheral neuropathy, CNS related disorders), gastroinstestinal disorders, Raynaud phenomenon, and pericarditis. In a preferred embodiment, therapeutic and pharmaceutical compositions of the invention are used to treat, prevent, ameliorate, diagnose, or prognose, renal disorders associated with systemic lupus erythematosus. In a most preferred embodiment, therapeutic and pharmaceutical compositions of the invention are used to treat, prevent, ameliorate, diagnose, or prognose, nephritis associated with systemic lupus erythematosus. In another most preferred embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate lupus or glomerular nephritis.

In a further specific embodiment, antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent hemolytic anemia. For example, patients diagnosed with autoimmune hemolytic anemia (AIHA), e.g., cryoglobinemia or Coombs positive anemia, are treated with an antibody, or antibodies, of the present invention. AIHA is an acquired hemolytic anemia due to auto-antibodies that react with the patient's red blood cells. The patient treated preferably will not have a B cell malignancy. Further adjunct therapies (such as glucocorticoids, prednisone, azathioprine, cyclophosphamide, vinca-laden platelets or Danazol) may be combined with the antibody therapy, but preferably the patient is treated with an antibody, or antibodies, of the present invention as a single-agent throughout the course of therapy. Antibodies of the present invention are administered to the hemolytic anemia patient according to a dosing schedule as described infra, which may be readily determined by one of ordinary skill in the art. Overall response rate is determined based upon an improvement in blood counts, decreased requirement for transfusions, improved hemoglobin levels and/or a decrease in the evidence of hemolysis as determined by standard chemical parameters. Administration of an antibody, or antibodies of the present invention will improve any one or more of the symptoms of hemolytic anemia in the patient treated as described above. For example, the patient treated as described above will show an increase in hemoglobin and an improvement in chemical parameters of hemolysis or return to normal as measured by serum lactic dehydrogenase and/or bilirubin.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Sjögren's Syndrome and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, HIV infection and/or medical conditions associated therewith (e.g. AIDS).

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Myasthenia gravis and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, IgA nephropathy and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, hemolytic anemia and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, thyroiditis and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Goodpasture's Syndrome and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, multiple sclerosis and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, chronic lymphocytic leukemia (CLL) and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, multiple myeloma and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Non-Hodgkin's lymphoma and/or medical conditions associated therewith.

In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Hodgkin's disease and/or medical conditions associated therewith.

In another specific embodiment, antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent adult immune thrombocytopenic purpura. Adult immune thrombocytopenic purpura (ITP) is a relatively rare hematologic disorder that constitutes the most common of the immune-mediated cytopenias. The disease typically presents with severe thrombocytopenia that may be associated with acute hemorrhage in the presence of normal to increased megakaryocytes in the bone marrow. Most patients with ITP have an IgG antibody directed against target antigens on the outer surface of the platelet membrane, resulting in platelet sequestration in the spleen and accelerated reticuloendothelial destruction of platelets (Bussell, J. B. Hematol. Oncol. Clin. North Am. (4):179 (1990)). A number of therapeutic interventions have been shown to be effective in the treatment of ITP. Steroids are generally considered first-line therapy, after which most patients are candidates for intravenous immunoglobulin (IVIG), splenectomy, or other medical therapies including vincristine or immunosuppressive/cytotoxic agents. Up to 80% of patients with ITP initially respond to a course of steroids, but far fewer have complete and lasting remissions. Splenectomy has been recommended as standard second-line therapy for steroid failures, and leads to prolonged remission in nearly 60% of cases yet may result in reduced immunity to infection. Splenectomy is a major surgical procedure that may be associated with substantial morbidity (15%) and mortality (2%). IVIG has also been used as second line medical therapy, although only a small proportion of adult patients with ITP achieve remission. Therapeutic options that would interfere with the production of autoantibodies by activated B cells without the associated morbidities that occur with corticosteroids and/or splenectomy would provide an important treatment approach for a proportion of patients with ITP. Patients with clinical diagnosis of ITP are treated with an antibody, or antibodies of the present invention, optionally in combination with steroid therapy. The patient treated will not have a B cell malignancy. Antibodies of the present invention are administered to the RA patient according to a dosing schedule as described infra, which may be readily determined by one of ordinary skill in the art. Overall patient response rate is determined based upon a platelet count determined on two consecutive occasions two weeks apart following treatments as described above. See, George et al. “Idiopathic Thrombocytopenic Purpura: A Practice Guideline Developed by Explicit Methods for The American Society of Hematology”, Blood 88:3-40 (1996), expressly incorporated herein by reference.

In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate an IgE-mediated allergic reaction or histamine-mediated allergic reaction. Examples of allergic reactions include, but are not limited to, asthma, rhinitis, eczema, chronic urticaria, and atopic dermatitis. In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent, or ameliorate anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility. In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate or modulate inflammation or an inflammatory disorder. Examples of chronic and acute inflammatory disorders that may be treated prevented or ameliorated with the therapeutic and pharmaceutical compositions of the invention include, but are not limited to, chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, Crohn's disease, inflammatory bowel disease, chronic and acute inflammatory pulmonary diseases, bacterial infection, psoriasis, septicemia, cerebral malaria, arthritis, gastroenteritis, and glomerular nephritis.

In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate ischemia and arteriosclerosis. Examples of such disorders include, but are not limited to, reperfusion damage (e.g., in the heart and/or brain) and cardiac hypertrophy.

Therapeutic or pharmaceutical compositions of the invention, may also be administered to modulate blood clotting and to treat or prevent blood clotting disorders, such as, for example, antibody-mediated thrombosis (i.e., antiphospholipid antibody syndrome (APS)). For example, therapeutic or pharmaceutical compositions of the invention, may inhibit the proliferation and differentiation of cells involved in producing anticardiolipin antibodies. These compositions of the invention can be used to treat, prevent, ameliorate, diagnose, and/or prognose thrombotic related events including, but not limited to, stroke (and recurrent stroke), heart attack, deep vein thrombosis, pulmonary embolism, myocardial infarction, coronary artery disease (e.g., antibody-mediated coronary artery disease), thrombosis, graft reocclusion following cardiovascular surgery (e.g., coronary arterial bypass grafts, recurrent fetal loss, and recurrent cardiovascular thromboembolic events.

Therapeutic or pharmaceutical compositions of the invention, may also be administered to treat, prevent, or ameliorate organ rejection or graft-versus-host disease (GVHD) and/or conditions associated therewith. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of antibodies of the invention, that inhibit an immune response, may be an effective therapy in preventing organ rejection or GVHD.

In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate a disease or disorder diseases associated with increased apoptosis including, but not limited to, AIDS, neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration), myelodysplastic syndromes (such as aplastic anemia), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia. In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate bone marrow failure, for example, aplastic anemia and myelodysplastic syndrome.

In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate growth, progression, and/or metastases of malignancies and proliferative disorders associated with increased cell survival, or the inhibition of apoptosis. Examples of such disorders, include, but are not limited to, leukemia (e.g., acute leukemia such as acute lymphocytic leukemia and acute myelocytic leukemia), neoplasms, tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma), heavy chain disease, metastases, or any disease or disorder characterized by uncontrolled cell growth.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used to treat or prevent a disorder characterized by hpergammagloulinemia (e.g., AIDS, autoimmune diseases, and some immunodeficiencies).

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used to treat or prevent a disorder characterized by deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, therapeutic or pharmaceutical compositions of the invention may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii.

Therapeutic or pharmaceutical compositions of the invention of the invention thereof, may be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith: primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.

Additionally, therapeutic or pharmaceutical compositions of the invention may be used to diagnose, prognose, treat or prevent one or more of the following diseases, disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia (e.g., anemia associated with parvovirus B19, patients with stable multiple myeloma who are at high risk for infection (e.g., recurrent infection), autoimmune hemolytic anemia (e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia (e.g., neonatal thrombocytopenia), and immune-mediated neutropenia), transplantation (e.g., cytomegalovirus (CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with risk factor for infection or morbidity), epilepsy (e.g., intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis (e.g., decompensation in myasthenia gravis), dermatomyositis, and polymyositis.

Additional preferred embodiments of the invention include, but are not limited to, the use of therapeutic or pharmaceutical compositions of the invention in the following applications:

Administration to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response. In a specific nonexclusive embodiment, therapeutic or pharmaceutical compositions of the invention are administered to boost the immune system to produce increased quantities of IgG. In another specific nonexclusive embodiment, antibodies of the are administered to boost the immune system to produce increased quantities of IgA. In another specific nonexclusive embodiment antibodies of the invention are administered to boost the immune system to produce increased quantities of IgM.

Administration to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741).

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a vaccine adjuvant that enhances immune responsiveness to specific antigen. In a specific embodiment, the vaccine is an antibody described herein. In another specific embodiment, the vaccine adjuvant is a polynucleotide described herein (e.g., an antibody polynucleotide genetic vaccine adjuvant). As discussed herein, therapeutic or pharmaceutical compositions of the invention may be administered using techniques known in the art, including but not limited to, liposomal delivery, recombinant vector delivery, injection of naked DNA, and gene gun delivery.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an adjuvant to enhance tumor-specific immune responses.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include, but are not limited to, virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and B, Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpes simplex, and yellow fever. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to the HIV gp120 antigen.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Neisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium (malaria).

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria).

In a specific embodiment, compositions of the invention may be administered to patients as vaccine adjuvants. In a further specific embodiment, compositions of the invention may be administered as vaccine adjuvants to patients suffering from an immune-deficiency. In a further specific embodiment, compositions of the invention may be administered as vaccine adjuvants to patients suffering from HIV.

In a specific embodiment, compositions of the invention may be used to increase or enhance antigen-specific antibody responses to standard and experimental vaccines. In a specific embodiment, compositions of the invention may be used to enhance seroconversion in patients treated with standard and experimental vaccines. In another specific embodiment, compositions of the invention may be used to increase the repertoire of antibodies recognizing unique epitopes in response to standard and experimental vaccination.

In a preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance antigen-specific antibody responses to standard and experimental vaccines by regulating binding of the soluble form of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP_(—)001183; and/or BAFF-R—GenBank accession number NP_(—)443177). In another preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance antigen-specific antibody responses to standard and experimental vaccines by regulating binding of the soluble form of APRIL to an APRIL receptor (e.g., BCMA and TACI).

In a preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance seroconversion in patients treated with standard and experimental vaccines by regulating binding of the soluble form of B Lymphocyte Stimulator to B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP_(—)001183; and/or BAFF-R—GenBank accession number NP_(—)443177). In another preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance seroconversion in patients treated with standard and experimental vaccines by regulating binding of the soluble form of APRIL to an APRIL receptor (e.g., BCMA and TACI).

In a preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance the repertoire of antibodies recognizing unique epitopes in response to standard and experimental vaccination by regulating binding of the soluble form of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP_(—)001183; and/or BAFF-R—GenBank accession number NP_(—)443177). In another preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance the repertoire of antibodies recognizing unique epitopes in response to standard and experimental vaccination by regulating binding of the soluble form of APRIL to an APRIL receptor (e.g., BCMA and TACI).

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a stimulator of B cell responsiveness to pathogens.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to induce higher affinity antibodies.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to increase serum immunoglobulin concentrations.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to accelerate recovery of immunocompromised individuals.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to boost immunoresponsiveness among aged populations.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy. B cell immunodeficiencies that may be ameliorated or treated by administering the antibodies and/or compositions of the invention include, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency.

In a specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate selective IgA deficiency.

In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate ataxia-telangiectasia.

In another specific embodiment antibodies and/or compositions of the invention are administered to treat or ameliorate common variable immunodeficiency.

In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate X-linked agammaglobulinemia.

In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate severe combined immunodeficiency (SCID).

In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate X-linked Ig deficiency with hyper IgM.

As an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering antibodies and/or compositions of the invention include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering antibodies and/or compositions of the invention include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, recovery from surgery.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a regulator of antigen presentation by monocytes, dendritic cells, T cells and/or B-cells. In one embodiment, antibody polypeptides or polynucleotides enhance antigen presentation or antagonize antigen presentation in vitro or in vivo. Moreover, in related embodiments, this enhancement or antagonization of antigen presentation may be useful in anti-tumor treatment or to modulate the immune system.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a mediator of mucosal immune responses. The expression of B Lymphocyte Stimulator on monocytes, the expression of B Lymphocyte Stimulator receptor on B cells, and the responsiveness of B cells to B Lymphocyte Stimulator suggests that it may be involved in exchange of signals between B cells and monocytes or their differentiated progeny. This activity is in many ways analogous to the CD40-CD154 signalling between B cells and T cells. Anti-B Lymphocyte Stimulator antibodies and compositions of the invention may therefore be good regulators of T cell independent immune responses to environmental pathogens. In particular, the unconventional B cell populations (CD5+) that are associated with mucosal sites and responsible for much of the innate immunity in humans may respond to antibodies or compositions of the invention thereby enhancing or inhibiting individual's immune status.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly, their susceptibility profile would likely change.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a monocyte cell specific binding protein to which specific activators or inhibitors of cell growth may be attached. The result would be to focus the activity of such activators or inhibitors onto normal, diseased, or neoplastic B cell populations.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a B cell specific binding protein to which specific activators or inhibitors of cell growth may be attached. The result would be to focus the activity of such activators or inhibitors onto normal, diseased, or neoplastic B cell populations.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means of detecting monocytic cells by virtue of its specificity. This application may require labeling the protein with biotin or other agents (e.g., as described herein) to afford a means of detection.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means of detecting B-lineage cells by virtue of its specificity. This application may require labeling the protein with biotin or other agents (e.g., as described herein) to afford a means of detection.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable immunodeficiency.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as part of a monocyte selection device the function of which is to isolate monocytes from a heterogeneous mixture of cell types. Antibodies of the invention could be coupled to a solid support to which monocytes would then specifically bind. Unbound cells would be washed out and the bound cells subsequently eluted. A non-limiting use of this selection would be to allow purging of tumor cells from, for example, bone marrow or peripheral blood prior to transplant.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as part of a B cell selection device the function of which is to isolate B cells from a heterogeneous mixture of cell types. Antibodies of the invention (that do not inhibit B Lymphocyte Stimulator/B Lymphocyte Stimulator Receptor interaction) binding soluble B Lymphocyte Stimulator could be coupled to a solid support to which B cells would then specifically bind. Unbound cells would be washed out and the bound cells subsequently eluted. A non-limiting use of this selection would be to allow purging of tumor cells from, for example, bone marrow or peripheral blood prior to transplant.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetence such as observed among SCID patients.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an antigen for the generation of antibodies to inhibit or enhance B Lymphocyte Stimulator mediated responses.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as pretreatment of bone marrow samples prior to transplant. Such treatment would increase B cell representation and thus accelerate recovery.

In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means of regulating secreted cytokines that are elicited by B Lymphocyte Stimulator and/or B Lymphocyte Stimulator receptor.

Antibody polypeptides or polynucleotides of the invention may be used to modulate IgE concentrations in vitro or in vivo.

Additionally, antibody polypeptides or polynucleotides of the invention may be used to treat, prevent, and/or diagnose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema.

In a specific embodiment, antibody polypeptides or polynucleotides of the invention, are administered to treat, prevent, diagnose, and/or ameliorate selective IgA deficiency.

In another specific embodiment antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate ataxia-telangiectasia.

In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate common variable immunodeficiency.

In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate X-linked agammaglobulinemia.

In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate severe combined immunodeficiency (SCID).

In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate X-linked Ig deficiency with hyper IgM. In a specific embodiment antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate X-linked Ig deficiency with hyper IgM.

In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, and/or diagnose chronic myelogenous leukemia, acute myelogenous leukemia, leukemia, hystiocytic leukemia, monocytic leukemia (e.g., acute monocytic leukemia), leukemic reticulosis, Shilling Type monocytic leukemia, and/or other leukemias derived from monocytes and/or monocytic cells and/or tissues.

In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate monocytic leukemoid reaction, as seen, for example, with tuberculosis.

In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate monocytic leukocytosis, monocytic leukopenia, monocytopenia, and/or monocytosis.

In a specific embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, detect, and/or diagnose monocyte disorders and/or diseases, and/or conditions associated therewith.

In a specific embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, detect, and/or diagnose primary B lymphocyte disorders and/or diseases, and/or conditions associated therewith. In one embodiment, such primary B lymphocyte disorders, diseases, and/or conditions are characterized by a complete or partial loss of humoral immunity. Primary B lymphocyte disorders, diseases, and/or conditions associated therewith that are characterized by a complete or partial loss of humoral immunity and that may be prevented, treated, detected and/or diagnosed with compositions of the invention include, but are not limited to, X-Linked Agammaglobulinemia (XLA), severe combined immunodeficiency disease (SCID), and selective IgA deficiency.

In a preferred embodiment antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose diseases or disorders affecting or conditions associated with any one or more of the various mucous membranes of the body. Such diseases or disorders include, but are not limited to, for example, mucositis, mucoclasis, mucocolitis, mucocutaneous leishmaniasis (such as, for example, American leishmaniasis, leishmaniasis americana, nasopharyngeal leishmaniasis, and New World leishmaniasis), mucocutaneous lymph node syndrome (for example, Kawasaki disease), mucoenteritis, mucoepidermoid carcinoma, mucoepidermoid tumor, mucoepithelial dysplasia, mucoid adenocarcinoma, mucoid degeneration, myxoid degeneration; myxomatous degeneration; myxomatosis, mucoid medial degeneration (for example, cystic medial necrosis), mucolipidosis (including, for example, mucolipidosis I, mucolipidosis II, mucolipidosis III, and mucolipidosis IV), mucolysis disorders, mucomembranous enteritis, mucoenteritis, mucopolysaccharidosis (such as, for example, type I mucopolysaccharidosis (i.e., Hurler's syndrome), type IS mucopolysaccharidosis (i.e., Scheie's syndrome or type V mucopolysaccharidosis), type II mucopolysaccharidosis (i.e., Hunter's syndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's syndrome), type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VI mucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VII mucopolysaccharidosis (i.e, mucopolysaccharidosis due to beta-glucuronidase deficiency), and mucosulfatidosis), mucopolysacchariduria, mucopurulent conjunctivitis, mucopus, mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virus diarrhea), mucous colitis (such as, for example, mucocolitis and myxomembranous colitis), and mucoviscidosis (such as, for example, cystic fibrosis, cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis). In a highly preferred embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose mucositis, especially as associated with chemotherapy.

In a preferred embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose diseases or disorders affecting or conditions associated with sinusitis.

An additional condition, disease or symptom that can be treated, prevented, and/or diagnosed by antibody polypeptides or polynucleotides of the invention is osteomyelitis.

An additional condition, disease or symptom that can be treated, prevented, and/or diagnosed by antibody polypeptides or polynucleotides of the invention is endocarditis.

All of the above described applications as they may apply to veterinary medicine.

Antibody polypeptides or polynucleotides of the invention may be used to treat, prevent, and/or diagnose diseases and disorders of the pulmonary system (e.g., bronchi such as, for example, sinopulmonary and bronchial infections and conditions associated with such diseases and disorders and other respiratory diseases and disorders. In specific embodiments, such diseases and disorders include, but are not limited to, bronchial adenoma, bronchial asthma, pneumonia (such as, e.g., bronchial pneumonia, bronchopneumonia, and tuberculous bronchopneumonia), chronic obstructive pulmonary disease (COPD), bronchial polyps, bronchiectasia (such as, e.g., bronchiectasia sicca, cylindrical bronchiectasis, and saccular bronchiectasis), bronchiolar adenocarcinoma, bronchiolar carcinoma, bronchiolitis (such as, e.g., exudative bronchiolitis, bronchiolitis fibrosa obliterans, and proliferative bronchiolitis), bronchiolo-alveolar carcinoma, bronchitic asthma, bronchitis (such as, e.g., asthmatic bronchitis, Castellani's bronchitis, chronic bronchitis, croupous bronchitis, fibrinous bronchitis, hemorrhagic bronchitis, infectious avian bronchitis, obliterative bronchitis, plastic bronchitis, pseudomembranous bronchitis, putrid bronchitis, and verminous bronchitis), bronchocentric granulomatosis, bronchoedema, bronchoesophageal fistula, bronchogenic carcinoma, bronchogenic cyst, broncholithiasis, bronchomalacia, bronchomycosis (such as, e.g., bronchopulmonary aspergillosis), bronchopulmonary spirochetosis, hemorrhagic bronchitis, bronchorrhea, bronchospasm, bronchostaxis, bronchostenosis, Biot's respiration, bronchial respiration, Kussmaul respiration, Kussmaul-Kien respiration, respiratory acidosis, respiratory alkalosis, respiratory distress syndrome of the newborn, respiratory insufficiency, respiratory scleroma, respiratory syncytial virus, and the like.

In a specific embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose chronic obstructive pulmonary disease (COPD).

In another embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose fibroses and conditions associated with fibroses, including, but not limited to, cystic fibrosis (including such fibroses as cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis), endomyocardial fibrosis, idiopathic retroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, pericentral fibrosis, perimuscular fibrosis, pipestem fibrosis, replacement fibrosis, subadventitial fibrosis, and Symmers' clay pipestem fibrosis.

In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate infectious diseases. Infectious diseases include diseases associated with yeast, fungal, viral and bacterial infections. Viruses causing viral infections which can be treated or prevented in accordance with this invention include, but are not limited to, retroviruses (e.g., human T-cell lymphotrophic virus (HTLV) types I and II and human immunodeficiency virus (HIV)), herpes viruses (e.g., herpes simplex virus (HSV) types I and II, Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), arenavirues (e.g., lassa fever virus), paramyxoviruses (e.g., morbillivirus virus, human respiratory syncytial virus, mumps, and pneumovirus), adenoviruses, bunyaviruses (e.g., hantavirus), cornaviruses, filoviruses (e.g., Ebola virus), flaviviruses (e.g., hepatitis C virus (HCV), yellow fever virus, and Japanese encephalitis virus), hepadnaviruses (e.g., hepatitis B viruses (HBV)), orthomyoviruses (e.g., influenza viruses A, B and C), papovaviruses (e.g., papillomavirues), picornaviruses (e.g., rhinoviruses, enteroviruses and hepatitis A viruses), poxviruses, reoviruses (e.g., rotavirues), togaviruses (e.g., rubella virus), rhabdoviruses (e.g., rabies virus). Microbial pathogens causing bacterial infections include, but are not limited to, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp., and Helicobacter pylori.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of B Lymphocyte Stimulator and/or its receptor, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises, or alternatively consists of, nucleic acids encoding an antibody, said nucleic acids being part of an expression vector that expresses the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acids have promoters, preferably heterologous promoters, operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is an scFv; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments or variants thereof, of an antibody.

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention or fragments or variants thereof are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:29 1-302 (1994), which describes the use of a retroviral vector to deliver the mdr 1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Klein et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcellmediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Clin. Pharma. Ther. 29:69-92m (1985)) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody or fragment thereof are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 7 1:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

Demonstration of Therapeutic or Prophylactic Utility of a Composition

The compounds of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays which can be used to determine whether administration of a specific antibody or composition of the present invention is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered an antibody or composition of the present invention, and the effect of such an antibody or composition of the present invention upon the tissue sample is observed. In various specific embodiments, in vitro assays can be carried out with representative cells of cell types involved in a patient's disorder, to determine if an antibody or composition of the present invention has a desired effect upon such cell types. Preferably, the antibodies or compositions of the invention are also tested in in vitro assays and animal model systems prior to administration to humans.

Antibodies or compositions of the present invention for use in therapy can be tested for their toxicity in suitable animal model systems, including but not limited to rats, mice, chicken, cows, monkeys, and rabbits. For in vivo testing of an antibody or composition's toxicity any animal model system known in the art may be used.

Efficacy in treating or preventing viral infection may be demonstrated by detecting the ability of an antibody or composition of the invention to inhibit the replication of the virus, to inhibit transmission or prevent the virus from establishing itself in its host, or to prevent, ameliorate or alleviate the symptoms of disease a progression. The treatment is considered therapeutic if there is, for example, a reduction in viral load, amelioration of one or more symptoms, or a decrease in mortality and/or morbidity following administration of an antibody or composition of the invention.

Antibodies or compositions of the invention can be tested for the ability to induce the expression of cytokines such as IFN-γ, by contacting cells, preferably human cells, with an antibody or composition of the invention or a control antibody or control composition and determining the ability of the antibody or composition of the invention to induce one or more cytokines. Techniques known to those of skill in the art can be used to measure the level of expression of cytokines. For example, the level of expression of cytokines can be measured by analyzing the level of RNA of cytokines by, for example, RT-PCR and Northern blot analysis, and by analyzing the level of cytokines by, for example, immunoprecipitation followed by western blot analysis and ELISA. In a preferred embodiment, a compound of the invention is tested for its ability to induce the expression of IFN-γ.

Antibodies or compositions of the invention can be tested for their ability to modulate the biological activity of immune cells by contacting immune cells, preferably human immune cells (e.g., T-cells, B-cells, and Natural Killer cells), with an antibody or composition of the invention or a control compound and determining the ability of the antibody or composition of the invention to modulate (i.e, increase or decrease) the biological activity of immune cells. The ability of an antibody or composition of the invention to modulate the biological activity of immune cells can be assessed by detecting the expression of antigens, detecting the proliferation of immune cells (i.e., B-cell proliferation), detecting the activation of signaling molecules, detecting the effector function of immune cells, or detecting the differentiation of immune cells. Techniques known to those of skill in the art can be used for measuring these activities. For example, cellular proliferation can be assayed by ³H-thymidine incorporation assays and trypan blue cell counts. Antigen expression can be assayed, for example, by immunoassays including, but not limited to, competitive and non-competitive assay systems using techniques such as western blots, immunohistochemistry radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and FACS analysis. The activation of signaling molecules can be assayed, for example, by kinase assays and electrophoretic shift assays (EMSAs). In a preferred embodiment, the ability of an antibody or composition of the invention to induce B-cell proliferation is measured. In another preferred embodiment, the ability of an antibody or composition of the invention to modulate immunoglobulin expression is measured.

Antibodies or compositions of the invention can be tested for their ability to reduce tumor formation in in vitro, ex vivo and in vivo assays. Antibodies or compositions of the invention can also be tested for their ability to inhibit viral replication or reduce viral load in in vitro and in vivo assays. Antibodies or compositions of the invention can also be tested for their ability to reduce bacterial numbers in in vitro and in vivo assays known to those of skill in the art. Antibodies or compositions of the invention can also be tested for their ability to alleviate of one or more symptoms associated with cancer, an immune disorder (e.g., an inflammatory disease), a neurological disorder or an infectious disease. Antibodies or compositions of the invention can also be tested for their ability to decrease the time course of the infectious disease. Further, antibodies or compositions of the invention can be tested for their ability to increase the survival period of animals suffering from disease or disorder, including cancer, an immune disorder or an infectious disease. Techniques known to those of skill in the art can be used to analyze the function of the antibodies or compositions of the invention in vivo.

Therapeutic/Prophylactic Compositions and Administration

The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of antibody (or fragment or variant thereof) or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, an antibody or fragment or variant thereof is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to, animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably a human.

Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin as described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

Various delivery systems are known and can be used to administer antibody or fragment or variant thereof of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a preferred embodiment the antibody of the invention is formulated in 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, 0.01% polysorbate 80, pH 6.5 (±0.3). In another preferred embodiment, the antibody of the invention is formulated in 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, 0.01% polysorbate 80, pH 6.5 (±0.3) for intravenous administration.

In a preferred embodiment the antibody of the invention is formulated in 110 mM sodium citrate, 8% sucrose, 0.04% (w/v) polysorbate 80 (pH 6.5) (±0.3). In another preferred embodiment, the antibody of the invention is formulated in 10 mM sodium citrate, 8% sucrose, 0.04% (w/v) polysorbate 80 (pH 6.5) for intravenous administration. In another preferred embodiment, the antibody of the invention is formulated in 10 mM sodium citrate, 8% sucrose, 0.04% (w/v) polysorbate 80 (pH 6.5) for subcutaneous administration.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

In another embodiment, the composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In yet another embodiment, the composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:20 1 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:35 1 (1989); Howard et al., J. Neurosurg. 7 1:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment where the composition of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of an antibody or a fragment thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the composition of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. In preferred embodiments, a dose of 1, 4, 10, or 20 mg/kg is administered intravenously to a patient. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of therapeutic or pharmaceutical compositions of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

The antibodies and antibody compositions of the invention may be administered alone or in combination with other adjuvants. Adjuvants that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, antibody and antibody compositions of the invention are administered in combination with alum. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis, and/or PNEUMOVAX-23™. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

In another specific embodiment, antibody and antibody compositions of the invention are used in combination with PNEUMOVAX-23™ to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated therewith. In one embodiment, antibody and antibody compositions of the invention are used in combination with PNEUMOVAX-23™ to treat, prevent, and/or diagnose any Gram positive bacterial infection and/or any disease, disorder, and/or condition associated therewith. In another embodiment, antibody and antibody compositions of the invention are used in combination with PNEUMOVAX-23™ to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated with one or more members of the genus Enterococcus and/or the genus Streptococcus. In another embodiment, antibody and antibody compositions of the invention are used in any combination with PNEUMOVAX-23™ to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated with one or more members of the Group B streptococci. In another embodiment, antibody and antibody compositions of the invention are used in combination with PNEUMOVAX-23™ to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated with Streptococcus pneumoniae.

The antibody and antibody compositions of the invention may be administered alone or in combination with other therapeutic agents, including but not limited to, chemotherapeutic agents, antibiotics, antivirals, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents and cytokines. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, the antibody and antibody compositions of the invention are administered in combination with other members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), TRAIL (also known as AIM-1, International Publication No. WO97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6):1185-1190 (1998)), endokine-alpha (International Publication No. WO 98/07880), Neutrokine-alpha (International Application Publication No. WO 98/18921), OPG, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.

In another embodiment, the compositions of the invention are administered in combination with Neutrokine-alpha receptors and/or Neurtokine-alpha SV receptors (e.g., TACI, BCMA and BAFF-R). In preferred in emodiments the Neutrokine-alpha receptors and/or Neurtokine-alpha SV receptors are soluble. In other preferred embodiments the Neutrokine-alpha receptors and/or Neurtokine-alpha SV receptors are fused to the FC region of an immunoglobulon molecule (e.g, amino acid residues 1-154 of TACI (GenBank accession number AAC51790), amino acids 1-48 of BCMA (GenBank accession number NP_(—)001183, or amino acids 1 to 81 of BAFF-R (GenBank Acession Number NP_(—)443177) fused to the Fc region of an IgG molecule.

In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with CD40 ligand (CD40L), a soluble form of CD40L (e.g., AVREND™), biologically active fragments, variants, or derivatives of CD40L, anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonistic or antagonistic antibodies).

In an additional embodiment, the antibody and antibody compositions of the invention are administered alone or in combination with an anti-angiogenic agent(s). Anti-angiogenic agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, Angiostatin (Entremed, Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, 1992); and metalloproteinase inhibitors such as BB94.

Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, (Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C. Storgard et al., J Clin. Invest. 103:47-54 (1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and 5-Fluorouracil.

Anti-angiogenic agents that may be administered in combination with the compositions of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, AG-3340 (Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg, Md.). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco, Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosan polysulfate (Georgetown University, Washington, D.C.).

In particular embodiments, the use of antibody and antibody compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.

In a particular embodiment, the use of antibody and antibody compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of antibody and antibody compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.

In particular embodiments, the use of antibody and antibody compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of strokes.

In another embodiment, antibody and antibody compositions of the invention are administered in combination with an anticoagulant. Anticoagulants that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, heparin, warfarin, and aspirin. In a specific embodiment, antibody and antibody compositions of the invention are administered in combination with heparin and/or warfarin. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with warfarin. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with heparin. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with heparin and aspirin.

In another embodiment, antibody and antibody compositions of the invention are administered in combination with an agent that suppresses the production of anticardiolipin antibodies. In specific embodiments, the polynucleotides of the invention are administered in combination with an agent that blocks and/or reduces the ability of anticardiolipin antibodies to bind phospholipid-binding plasma protein beta 2-glycoprotein I (b2GPI).

In certain embodiments, antibody and antibody compositions of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with antibody and antibody compositions of the invention to treat, prevent, and/or diagnose AIDS and/or to treat, prevent, and/or diagnose HIV infection.

In certain embodiments, antibody and antibody compositions of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the compositions of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administered in combination with the compositions of the invention, include, but are not limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with compositions of the invention to treat AIDS and/or to prevent or treat HIV infection.

Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosine NRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/FTC; structurally related to lamivudine (3TC) but with 3- to 10-fold greater activity in vitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related to lamivudine but retains activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused approval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; its active form is PMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG (active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC, with activity against AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3′azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl (SATE)-bearing prodrug forms of b-L-FD4C and b-L-FddC (WO 98/17281).

Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N mutation; DuPont); GW-420867X (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent from the latex tree; active against viruses containing either or both the Y181C and K103N mutations); and Propolis (WO 99/49830).

Additional protease inhibitors include LOPINAVIR™ (ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); and AGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting state and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a second-generation fusion inhibitor; Trimeris).

Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine receptor agonists such as RANTES, SDF-1, MIP-1a, MIP-1b, etc., may also inhibit fusion.

Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor; Arondex); and naphthols such as those disclosed in WO 98/50347.

Additional antiretroviral agents include hydroxyurea-like compounds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOX™ (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and myvopholic acids such as CellCept (mycophenolate mofetil; Roche).

Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear translocation such as arylene bis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.

Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-1a, MIP-1b, SDF-1a, IL-2, PROLEUKIN™ (aldesleukin/L2-7001; Chiron), IL-4, IL-8, IL-10, IL-12, and IL-13; interferons such as IFN-a2a; antagonists of TNFs, NFkB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003 (Apolion), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapies such as genetic suppressor elements (GSEs; WO 98/54366), and intrakines (genetically modified CC chemokines targetted to the ER to block surface expression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-a antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, and a-naphthoflavone (WO 98/30213); and antioxidants such as g-L-glutamyl-L-cysteine ethyl ester (g-GCE; WO 99/56764).

In other embodiments, antibody and antibody compositions of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, antibody and antibody compositions of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat, prevent, and/or diagnose an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium avium complex infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with GANCICLOVIR™ FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat, prevent, and/or diagnose an opportunistic cytomegalovirus infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with FLUCONAZOLE™ ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat, prevent, and/or diagnose an opportunistic fungal infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat, prevent, and/or diagnose an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat, prevent, and/or diagnose an opportunistic Toxoplasma gondii infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat, prevent, and/or diagnose an opportunistic bacterial infection.

In a further embodiment, the antibody and antibody compositions of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

In a further embodiment, the antibody and antibody compositions of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, amoxicillin, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents, that may be administered in combination with the compositions of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININ™), brequinar, deoxyspergualin, and azaspirane (SKF 105685).

In specific embodiments, antibody and antibody compositions of the invention are administered in combination with immunosuppressants Immunosuppressants preparations that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, ORTHOCLONE™ (OKT® 3 (muromonab-CD3), SANDIMMUNE™, NEORAL™ SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURAN™ (azathioprine), glucorticosteroids, adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™ (prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™ (methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with steroid therapy. Steroids that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, oral corticosteroids, prednisone, and methylprednisolone (e.g., IV methylprednisolone). In a specific embodiment, antibody and antibody compositions of the invention are administered in combination with prednisone. In a further specific embodiment, the antibody and antibody compositions of the invention are administered in combination with prednisone and an immunosuppressive agent Immunosuppressive agents that may be administered with the antibody and antibody compositions of the invention and prednisone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV. In a another specific embodiment, antibody and antibody compositions of the invention are administered in combination with methylprednisolone. In a further specific embodiment, the antibody and antibody compositions of the invention are administered in combination with methylprednisolone and an immunosuppressive agent Immunosuppressive agents that may be administered with the antibody and antibody compositions of the invention and methylprednisolone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.

In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with an antimalarial. Antimalarials that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with an NSAID.

In a nonexclusive embodiment, the antibody and antibody compositions of the invention are administered in combination with one, two, three, four, five, ten, or more of the following drugs: NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Dong Wha), darbufelone mesylate (Warner-Lambert), soluble TNF receptor 1 (synergen; Amgen), IPR-6001 (Institute for Pharmaceutical Research), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim), LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), and butixocort propionate (Warner Lambert).

In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: Infliximab (also known as Remicade™ Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (also known as Arava™ from Hoechst Marion Roussel), Kineret™ (an IL-1 Receptor antagonist also known as Anakinra from Amgen, Inc.), SCIO-469 (p38 kinase inhibitor from Scios, Inc), Humira® (adalimumab from Abbott Laboratories) and/or ASLERA™ (prasterone, dehydroepiandrosterone, GL701) from Genelabs Technologies Inc.

In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with one, two, three, four, five or more of the following drugs: methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g., as described herein), cyclophosphamide, chlorambucil, gold, ENBREL™ (Etanercept), anti-TNF antibody, LJP 394 (La Jolla Pharmaceutical Company, San Diego, Calif.) and prednisolone.

In a more preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with an antimalarial, methotrexate, anti-TNF antibody, ENBREL™ and/or suflasalazine. In one embodiment, the antibody and antibody compositions of the invention are administered in combination with methotrexate. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with anti-TNF antibody. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with methotrexate and anti-TNF antibody. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with suflasalazine. In another specific embodiment, the antibody and antibody compositions of the invention are administered in combination with methotrexate, anti-TNF antibody, and suflasalazine. In another embodiment, the antibody and antibody compositions of the invention are administered in combination ENBREL™. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with ENBREL™ and methotrexate. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with ENBREL™, methotrexate and suflasalazine. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with ENBREL™, methotrexate and suflasalazine. In other embodiments, one or more antimalarials is combined with one of the above-recited combinations. In a specfic embodiment, the antibody and antibody compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), ENBREL™, methotrexate and suflasalazine. In another specfic embodiment, the antibody and antibody compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNF antibody, and methotrexate.

In an additional embodiment, antibody and antibody compositions of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the antibody and antibody compositions of the invention include, but not limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, and GAMIMUNE™. In a specific embodiment, antibody and antibody compositions of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the antibody and antibody compositions of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

In specific embodiments, the compositions of the invention are administered alone or in combination with anti-CD4 antibody. In one embodiment, coadministration of the compositions of the invention with anti-CD4 antibody is envisoned for treatment of rheumatoid arthritis.

In specific embodiments, the compositions of the invention are administered alone or in combination with anti-IL-15 antibody. In one embodiment, coadministration of the compositions of the invention with anti-IL-15 antibody is envisoned for treatment of rheumatoid arthritis.

In specific embodiments, the compositions of the invention are administered alone or in combination with CTLA4-Ig and LEA29Y. In one embodiment, coadministration of the compositions of the invention with CTLA4-Ig and LEA29Y is envisoned for treatment of rheumatoid arthritis.

In specific embodiments, the compositions of the invention are administered alone or in combination with anti-IL-6 Receptor antibody. In one embodiment, coadministration of the compositions of the invention with anti-IL-6 Receptor antibody is envisoned for treatment of rheumatoid arthritis.

In specific embodiments, the compositions of the invention are administered alone or in combination with anti-C5 (complement component) antibody. In one embodiment, coadministration of the compositions of the invention with anti-C5 antibody is envisoned for treatment of rheumatoid arthritis.

In specific embodiments, the compositions of the invention are administered alone or in combination with complement cascade inhibitors. Complement cascade inhibitors include, but are notlimited to, anti-properdin antibodies (Gliatech); TP-10, a recombinant soluble type I complement receptor (AVANT Immunotheragenetics Inc.); Pexelizmab, a Complement C5 inhibitor (Alexion Pharmaceuticals Inc.); and 5G1.1, a monoclonal antibody that prevents cleavage of complement component C5 into its pro-inflammatory components. In one embodiment, coadministration of the compositions of the invention with complement cascade inhibitors are is envisoned for treatment of Inflammation, Rheumatoid arthritis, and/or cardiovascular disorders.

In another embodiment, compostions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab (anti-CD20 antibody from Coulter Pharmaceuticals, San Francisco, Calif.). In a further embodiment, compositions of the invention are administered with tositumomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. Tositumomab may optionally be associated with 131I. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.

In another specific embodiment, the compositions of the invention are administered in combination Zevalin™. In a further embodiment, compositions of the invention are administered with Zevalin™ and CHOP, or Zevalin™ and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may be associated with one or more radisotopes. Particularly preferred isotopes are 90Y and 111In.

In additional preferred embodiments, compositions of the invention are administered in combination with Rituximab (Rituxan™) and/or Ibritumomab Tiuxetan (Zevalin™, e.g., either (In-111) Ibritumomab Tiuxetan or (Y-90) Ibritumomab Tiuxetan). In a specific embodiment, compositions of the invention are administered in combination with Rituximab and/or Ibritumomab Tiuxetan for the treatment of non-Hodgkin's lymphoma.

In additional preferred embodiments, compositions of the invention are administered in combination with imatinib mesylate (Gleevec®: 4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate). In a specific embodiment, compositions of the invention are administered in combination with imatinib mesylate for the treatment of chronic myelogenous leukemia.

In additional preferred embodiments, compositions of the invention are administered in combination with bortezomib (Velcade™ [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl) amino]propyl]amino]butyl]boronic acid). In a specific embodiment, compositions of the invention are administered in combination with bortezomib for the treatment of multiple myeloma.

In additional preferred embodiments, compositions of the invention are administered in combination with Alemtuzumab (Campath®). In a specific embodiment, compositions of the invention are administered in combination with Alemtuzumab for the treatment of chronic lymphocytic leukemia.

In additional preferred embodiments, compositions of the invention are administered in combination with fludarabine phosphate (Fludara®: 9H-Purin-6-amine, 2-fluoro-9-(5-O-phosphono-b-D-arabinofuranosyl) (2-fluoro-ara-AMP)). In a specific embodiment, compositions of the invention are administered in combination with fludarabine phosphate for the treatment of chronic lymphocytic leukemia.

In further particular embodiments, compositions of the present invention are used to treat, ameliorate and/or prevent hematological cancers. Compositions of the present invention may be used in combination with one or more surgical and/or radiological procedures and/or therapeutic agents to treat, ameliorate and/or prevent hematological cancers. Hematological cancers which may be treated using compositions of the present invention include, but are not limited to, non-Hodgkin's lymphoma (e.g., small lymphocytic lymphoma, follicular center cell lymphoma, lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle cell lymphoma, immunoblastic lymphoma, burkitt's lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma and intestinal T-cell lymphoma), leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and plasma cell neoplasms including multiple myeloma.

In preferred embodiments, compositions of the present invention are used to treat, ameliorate and/or prevent hematological cancers. compositions of the present invention may be used in combination with one or more surgical and/or radiological procedures and/or therapeutic agents to treat, ameliorate and/or prevent hematological cancers. Hematological cancers which may be treated using compositions of the present invention include, but are not limited to, non-Hodgkin's lymphoma (e.g., small lymphocytic lymphoma, follicular center cell lymphoma, lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle cell lymphoma, immunoblastic lymphoma, burkitt's lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma and intestinal T-cell lymphoma), leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and plasma cell neoplasms including multiple myeloma.

In one preferred embodiment, compositions of the present invention are used to treat plasma cell neoplasms. In a specific embodiment, that plasma cell neoplasm is multiple myeloma.

In another preferred embodiment, compositions of the present invention are used to treat non-Hodgkin's lymphoma.

In another preferred embodiment, compositions of the present invention are used to treat leukemia. In a specific embodiment, that leukemia is acute lymphocytic leukemia. In another specific embodiment, that leukemia is chronic lymphocytic leukemia.

Compositions of the present invention may be administered in combination with one or more surgical and/or radiological procedures useful in the treatment of hematological cancer including, but not limited to, bone marrow transplantation, external beam radiation and total body irradiation.

In preferred embodiments, compositions of the present invention are administered in combination with one or more surgical and/or radiological procedures useful in the treatment of hematological cancer including, but not limited to, bone marrow transplantation, external beam radiation and total body irradiation.

In one preferred embodiment, compositions of the present invention may be administered in combination with one or more surgical and/or radiological procedures useful in the treatment of multiple myeloma including, but not limited to, allogeneic bone marrow transplantation and peripheral stem cell support.

In another preferred embodiment, compositions of the present invention may be administered in combination with one or more surgical and/or radiological procedures useful in the treatment of non-Hodgkin's lymphoma including, but not limited to, allogeneic bone marrow transplantation and peripheral stem cell support.

In further specific embodiments, compositions of the present invention may be administered in combination with one or more surgical and/or radiological procedures useful in the treatment of leukemia including, but not limited to, allogeneic bone marrow transplantation and peripheral stem cell support. In one specific preferred embodiment, compositions of the present invention of the invention are used to treat acute lymphocytic leukemia (ALL). In another specific preferred embodiment, compositions of the present invention are used to treat chronic lymphocytic leukemia (CLL).

Compositions of the present invention may be administered in combination with one or more therapeutic agents useful in the treatment of multiple myeloma including, but not limited to, Alkylating agents, Anthracyclines, Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), Cyclophosphamide (Cytoxan®, Neosar®, CTX), Dexamethasone (Decadron®), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Melphalan (L-PAM, Alkeran®, Phenylalanine mustard), Prednisone, Thalidomide and Vincristine (Oncovorin®, Onco TCS®, VCR, Leurocristine®).

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of multiple myeloma.

Preferred combinations of therapeutic agents useful in the treatment of multiple myeloma which may be administered in combination with compositions of the present invention include, but are not limited to, Cyclophosphamide+Prednisone, Melphalan+Prednisone (MP), Vincristine+Adriamycin®+Dexamethasone (VAD), Vincristine+Carmustine+Melphalan+Cyclophosphamide+Prednisone (VBMCP; the M2 protocol), and Vincristine+Melphalan+Cyclophosphamide+Prednisone alternating with Vincristine+Carmustine+Doxorubicin+Prednisone (VMCP/VBAP).

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agent combinations in the treatment, amelioration and/or prevention of multiple myeloma.

Compositions of the present invention may be administered in combination with one or more therapeutic agents useful in the treatment of non-Hodgkin's lymphoma including, but not limited to, 2-chlorodeoxyadenosine, Amifostine (Ethyol®, Ethiofos®, WR-272), Bexarotene (Targretin®, Targretin Gel®, Targretin Oral®, LGD1069), Bleomycin (Blenoxane®), Busulfan (Busulfex®, Myleran®), Carboplatin (Paraplatin®, CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), Chlorambucil (Leukeran®), Cisplatin (Platinol®, CDDP), Cladribine (2-CdA, Leustatin®), Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®), Dacarbazine (DTIC), Daunorubicin (Daunomycin, DaunoXome®, Daunorubicin®, Cerubidine®), Denileukin diftitox (Ontak®), Dexamethasone (Decadron®), Dolasetron mesylate (Anzemet®), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Erythropoietin (EPO®, Epogen®, Procrit®), Etoposide phosphate (Etopophos®), Etoposide (VP-16, Vepesid®), Fludarabine (Fludara®, FAMP), Granisetron (Kytril®), Hydrocortisone, Idarubicin (Idamycin®, DMDR, IDA), Ifosfamide (IFEX®), Interferon alpha (Alfaferone®, Alpha-IF®), Interferon alpha 2a (Intron A®), Mechlorethamine (Nitrogen Mustard, HN2, Mustargen®), Melphalan (L-PAM, Alkeran®, Phenylalanine mustard), Methotrexate® (MTX, Mexate®, Folex®), Methylprednisolone (Solumedrol®), Mitoxantrone (Novantrone®, DHAD), Ondansetron (Zofran®), Pentostatin (Nipent®, 2-deoxycoformycin), Perfosfamide (4-hydroperoxycyclophosphamide, 4-HC), Prednisone, Procarbazine (Matulane®), Rituximab® (Rituxan®, anti-CD20 MAb), Thiotepa (triethylenethiophosphaoramide, Thioplex®), Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®), Vinblastine (Velban®, VLB), Vincristine (Oncovin®, Onco TCS®, VCR, Leurocristine®) and Vindesine (Eldisine®, Fildesin®).

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of non-Hodgkin's lymphoma.

Preferred combinations of therapeutic agents useful in the treatment of non-Hodgkin's lymphoma which may be administered in combination with compositions of the present invention include, but are not limited to, Adriamycin®+Blenoxane+Vinblastine+Dacarbazine (ABVD), Anti-idiotype therapy (BsAb)+Interferon alpha, Anti-idiotype therapy (BsAb)+Chlorambucil, Anti-idiotype therapy (BsAb)+Interleukin-2, BCNU (Carmustine)+Etoposide+Ara-C (Cytarabine)+Melphalen (BEAM), Bleomycin+Etoposide+Adriamycin+Cyclophosphamide+

Vincristine+Procarbazine+Prednisone (BEACOPP), Bryostatin+Vincristine, Cyclophosphamide+BCNU (Carmustine)+VP-16 (Etoposide) (CBV), Cyclophosphamide+Vincristine+Prednisone (CVP), Cyclophosphamide+Adriamycin® (Hydroxyldaunomycin)+Vincristine (Oncovorin)+Prednisone (CHOP), Cyclophosphamide+Novantrone® (Mitoxantrone)+Vincristine (Oncovorin)+Prednisone (CNOP), Cyclophosphamide+Doxorubicin+Teniposide+Prednisone, Cyclophosphamide+Adriamycin® (Hydroxyldaunomycin)+Vincristine (Oncovorin)+Prednisone+Rituximab (CHOP+Rituximab), Cyclophosphamide+Doxorubicin+Teniposide+Prednisone+Interferon alpha, Cytarabine+Bleomycin+Vincristine+Methotrexate (CytaBOM), Dexamethasone+Cytarabine+Cisplatin (DHAP), Dexamethasone+Ifosfamide+Cisplatin+Etoposide (DICE), Doxorubicin+Vinblastine+Mechlorethamine+Vincristine+Bleomycin+Etoposide+Prednisone (Stanford V), Etoposide+Vinblastine+Adriamycin (EVA), Etoposide+Methylprednisone+Cytarabine+Cisplatin (ESHAP), Etoposide+Prednisone+Ifosfamide+Cisplatin (EPIC), Fludarabine, Mitoxantrone+Dexamethasone (FMD), Fludarabine, Dexamethasone, Cytarabine (ara-C), +Cisplatin (Platinol®) (FluDAP), Ifosfamide+Cisplatin+Etoposide (ICE), Mechlorethamine+Oncovin® (Vincristine)+Procarbazine+Prednisone (MOPP), Mesna+Ifosfamide+Idarubicin+Etoposide (MIZE), Methotrexate with leucovorin rescue+Bleomycin+Adriamycin+Cyclophosphamide+Oncovorin+Dexamethasone (m-BACOD), Prednisone+Methotrexate+Adriamycin+Cyclophosphamide+Etoposide (ProMACE), Thiotepa+Busulfan+Cyclophosphamide, Thiotepa+Busulfan+Melphalan, Topotecan+Paclitaxel, and Vincristine (Oncovin®)+Adriamycin®+Dexamethasone (VAD).

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agent combinations in the treatment, amelioration and/or prevention of non-Hodgkin's lymphoma.

Further examples of therapeutic agents useful in the treatment of non-Hodgkin's lymphoma which may be administered in combination with compositions of the present invention include, but are not limited to, A007 (4-4′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone), AG-2034 (AG-2024, AG-2032, GARFT [glycinamide ribonucleoside transformylase] inhibitor), Aldesleukin (IL-2, Proleukin®), Alemtuzumab (Campath®), Alitretinoin (Panretin®, LGN-1057), Altretamine (Hexylen®, hexamethylmelamine, Hexastat®), Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Anti-CD19/CD3 MAb (anti-CD19/CD3 scFv, anti-NHL MAb), Anti-idiotype therapy (BsAb), Arabinosylguanine (Ara-G, GW506U78), Arsenic trioxide (Trisenox®, ATO), B43-Genistein (anti-CD19 Ab/genistein conjugate), B7 antibody conjugates, Betathine (Beta-LT), B Lymphocyte Stimulator antagonists, Bryostatin-1 (Bryostatin®, BMY-45618, NSC-339555), CHML (Cytotropic Heterogeneous Molecular Lipids), Clofarabine (chloro-fluoro-araA), Daclizumab (Zenapax®), Depsipeptide (FR901228, FK228), Dolastatin-10 (DOLA-10, NSC-376128), Epirubicin (Ellence®, EPI, 4′ epi-doxorubicin), Epratuzumab (Lymphocide®, humanized anti-CD22, HAT), Fly3/flk2 ligand (Mobista®), G3139 (Genasense®, GentaAnticode®, Bcl-2 antisense), Hu1D10 (anti-HLA-DR MAb, SMART 1D10), HumaLYM (anti-CD20 MAb), Ibritumomab tiuxetan (Zevalin®), Interferon gamma (Gamma-interferon, Gamma 100®, Gamma-IF), Irinotecan (Camptosar®, CPT-11, Topotecin®, CaptoCPT-1), ISIS-2053, ISIS-3521 (PKC-alpha antisense), Lmb-2 immunotoxin (anti-CD25 recombinant immuno toxin, anti-Tac(Fv)-PE38), Leuvectin® (cytofectin+IL-2 gene, IL-2 gene therapy), Lym-1 (1314 LYM-1), Lymphoma vaccine (Genitope), Nelarabine (Compound 506, U78), Neugene compounds (Oncomyc-NG®, Resten-NG®, myc antisense), NovoMAb-G2 scFv (NovoMAb-G2 IgM), O6-benzylguanine (BG, Procept®), Oxaliplatin (Eloxatine®, Eloxatin®), Paclitaxel (Paxene®, Taxol®), Paclitaxel-DHA (Taxoprexin®), Peldesine (BCX-34, PNP inhibitor), Rebeccamycin and Rebeccamycin analogues, SCH-66336, Sobuzoxane (MST-16, Perazolin®), SU5416 (Semaxanib®, VEGF inhibitor), TER-286, Thalidomide, TNP-470 (AGM-1470), Tositumomab (Bexxar®), Valspodar (PSC 833), Vaxid (B-cell lymphoma DNA vaccine), Vinorelbine (Navelbine®), WF10 (macrophage regulator) and XR-9576 (XR-9351, P-glycoprotein/MDR inhibitor).

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of non-Hodgkin's lymphoma.

Compositions of the present invention may be administered in combination with one or more therapeutic agents useful in the treatment of acute lymphocytic leukemia including, but not limited to, Amsacrine, Carboplatin (Paraplatin®, CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), Cholecaliferol, Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®), Daunorubicin (Daunomycin, DaunoXome®, Daunorubicin®, Cerubidine®), Dexamethasone (Decadron®), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Etoposide (VP-16, Vepesid®), Filgrastam® (Neupogen®, G-CSF, Leukine®), Fludarabine (Fludara®, FAMP), Idarubicin (Idamycin®, DMDR, IDA), Ifosfamide (IFEX®), Imatinib mesylate (STI-571, Imatinib®, Glivec®, Gleevec®, Abl tyrosine kinase inhibitor), Interferon gamma (Gamma-interferon, Gamma 1000, Gamma-IF), L-asparaginase (Elspar®, Crastinin®, Asparaginase Medac®, Kidrolase®), Mercaptopurine (6-mercaptopurine, 6-MP), Methotrexate® (MTX, Mexate®, Folex®), Mitoxantrone (Novantrone®, DHAD), Pegaspargase® (Oncospar®), Prednisone, Retinoic acid, Teniposide (VM-26, Vumon®), Thioguanine (6-thioguanine, 6-TG), Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®), Tretinoin (Retin-A®, Atragen®, ATRA, Vesanoid®) and Vincristine (Oncovorin®, Onco TCS®, VCR, Leurocristine®).

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of acute lymphocytic leukemia.

Further examples of therapeutic agents useful in the treatment of acute lymphocytic leukemia which may be administered in combination with compositions of the present invention include, but are not limited to, Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aminopterin, Annamycin (AR-522, annamycin LF, Aronex®), Arabinosylguanine (Ara-G, GW506U78, Nelzarabine®), Arsenic trioxide (Trisenox®, ATO, Atrivex®), B43-Genistein (anti-CD19 Ab/genistein conjugate), B43-PAP (anti-CD19 Ab/pokeweed antiviral protein conjugate), Cordycepin, CS-682, Decitabine (5-aza-2′-deoxyytidine), Dolastatin-10 (DOLA-10, NSC-376128), G3139 (Genasense®, GentaAnticode®, Bcl-2 antisense), Irofulven (MGI-114, Ivofulvan, Acylfulvene analogue), MS-209, Phenylbutyrate, Quinine, TNP-470 (AGM-1470, Fumagillin), Trimetrexate (Neutrexin®), Troxacitabine (BCH-204, BCH-4556, Troxatyl®), UCN-01 (7-hydroxystaurosporine), WHI-P131 and WT1 Vaccine.

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of acute lymphocytic leukemia.

Preferred combinations of therapeutic agents useful in the treatment of acute lymphocytic leukemia which may be administered in combination with compositions of the present invention include, but are not limited to, Carboplatin+Mitoxantrone, Carmustine+Cyclophosphamide+Etoposide, Cytarabine+Daunorubicin, Cytarabine+Doxorubicin, Cytarabine+Idarubicin, Cytarabine+Interferon gamma, Cytarabine+L-asparaginase, Cytarabine+Mitoxantrone, Cytarabine+Fludarabine and Mitoxantrone, Etoposide+Cytarabine, Etoposide+Ifosfamide, Etoposide+Mitoxantrone, Ifosfamide+Etoposide+Mitoxantrone, Ifosfamide+Teniposide, Methotrexate+Mercaptopurine, Methotrexate+Mercaptopurine+Vincristine+Prednisone, Phenylbutyrate+Cytarabine, Phenylbutyrate+Etoposide, Phenylbutyrate+Topotecan, Phenylbutyrate+Tretinoin, Quinine+Doxorubicin, Quinine+Mitoxantrone+Cytarabine, Thioguanine+Cytarabine+Amsacrine, Thioguanine+Etoposide+Idarubicin, Thioguanine+Retinoic acid+Cholecaliferol, Vincristine+Prednisone, Vincristine+Prednisone and L-asparaginase, Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin, Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Filgrastim, Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Cyclophosphamide+Methotrexate, and Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Cyclophosphamide+Methotrexate+Filgrastim.

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agent combinations in the treatment, amelioration and/or prevention of acute lymphocytic leukemia.

Compositions of the present invention may be administered in combination with one or more therapeutic agents useful in the treatment of chronic lymphocytic leukemia including, but not limited to, Chlorambucil (Leukeran®), Cladribine (2-CdA, Leustatin®), Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®, cytarabine ocfosfate, ara-CMP), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Fludarabine (Fludara®, FAMP), Pentostatin (Nipent®, 2-deoxycoformycin), Prednisone and Vincristine (Oncovorin®, Onco TCS CD, VCR, Leurocristine®).

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of chronic lymphocytic leukemia.

Further examples of therapeutic agents useful in the treatment of chronic lymphocytic leukemia which may be administered in combination with compositions of the present invention include, but are not limited to, Alemtuzumab (Campath®), Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aminopterin, Annamycin (AR-522, annamycin LF, Aronex®), Arabinosylguanine (Ara-G, GW506U78, Nelzarabine®, Compound 506U78), Arsenic trioxide (Trisenox®, ATO, Atrivex®), Bryostatin-1 (Bryostatin®, BMY-45618, NSC-339555), CS-682, Dolastatin-10 (DOLA-10, NSC-376128), Filgrastim (Neupogen®, G-CSF, Leukine), Flavopiridol (NSC-649890, HMR-1275), G3139 (Genasense®, GentaAnticode®, Bcl-2 antisense), Irofulven (MGI-114, Ivofulvan, Acylfulvene analogue), MS-209, Phenylbutyrate, Rituximab® (Rituxan®, anti-CD20 MAb), Thalidomide, Theophylline, TNP-470 (AGM-1470, Fumagillin), UCN-01 (7-hydroxystaurosporine) and WHI-P131.

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of chronic lymphocytic leukemia.

Preferred combinations of therapeutic agents useful in the treatment of chronic lymphocytic leukemia which may be administered in combination with compositions of the present invention include, but are not limited to, Fludarabine+Prednisone, and Cyclophosphamide+Doxorubicin+Vincristine+Prednisone (CHOP).

In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agent combinations in the treatment, amelioration and/or prevention of chronic lymphocytic leukemia.

In an additional embodiment, the antibody and antibody compositions of the invention are administered in combination with cytokines. Cytokines that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta. In preferred embodiments, antibody and antibody compositions of the invention are administered with B Lymphocyte Stimulator (e.g., amino acids 134-285 of SEQ IF D NO:3228). In another embodiment, antibody and antibody compositions of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, and IL-22. In preferred embodiments, the antibody and antibody compositions of the invention are administered in combination with IL4 and IL10.

In one embodiment, the antibody and antibody compositions of the invention are administered in combination with one or more chemokines. In specific embodiments, the antibody and antibody compositions of the invention are administered in combination with an α(C×C) chemokine selected from the group consisting of gamma-interferon inducible protein-10 (γIP-10), interleukin-8 (IL-8), platelet factor-4 (PF4), neutrophil activating protein (NAP-2), GRO-α, GRO-β, GRO-γ, neutrophil-activating peptide (ENA-78), granulocyte chemoattractant protein-2 (GCP-2), and stromal cell-derived factor-1 (SDF-1, or pre-B cell stimulatory factor (PBSF)); and/or a β(CC) chemokine selected from the group consisting of: RANTES (regulated on activation, normal T expressed and secreted), macrophage inflammatory protein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2), monocyte chemotactic protein-3 (MCP-3), monocyte chemotactic protein-4 (MCP-4) macrophage inflammatory protein-1 gamma (MIP-1γ), macrophage inflammatory protein-3 alpha (MIP-3α), macrophage inflammatory protein-3 beta (MIP-3β), macrophage inflammatory protein-4 (MIP-4/DC-CK-1/PARC), eotaxin, Exodus, and I-309; and/or the γ(C) chemokine, lymphotactin.

In another embodiment, the antibody and antibody compositions of the invention are administered with chemokine beta-8, chemokine beta-1, and/or macrophage inflammatory protein-4. In a preferred embodiment, the antibody and antibody compositions of the invention are administered with chemokine beta-8.

In an additional embodiment, the antibody and antibody compositions of the invention are administered in combination with an IL-4 antagonist. IL-4 antagonists that may be administered with the antibody and antibody compositions of the invention include, but are not limited to: soluble IL-4 receptor polypeptides, multimeric forms of soluble IL-4 receptor polypeptides; anti-IL-4 receptor antibodies that bind the IL-4 receptor without transducing the biological signal elicited by IL-4, anti-IL4 antibodies that block binding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 that bind IL-4 receptors but do not transduce the biological signal elicited by IL-4. Preferably, the antibodies employed according to this method are monoclonal antibodies (including antibody fragments, such as, for example, those described herein).

The invention also encompasses combining the polynucleotides and/or polypeptides of the invention (and/or agonists or antagonists thereof) with other proposed or conventional hematopoietic therapies. Thus, for example, the polynucleotides and/or polypeptides of the invention (and/or agonists or antagonists thereof) can be combined with compounds that singly exhibit erythropoietic stimulatory effects, such as erythropoietin, testosterone, progenitor cell stimulators, insulin-like growth factor, prostaglandins, serotonin, cyclic AMP, prolactin, and triiodothyzonine. Also encompassed are combinations of the antibody and antibody compositions of the invention with compounds generally used to treat aplastic anemia, such as, for example, methenolene, stanozolol, and nandrolone; to treat iron-deficiency anemia, such as, for example, iron preparations; to treat malignant anemia, such as, for example, vitamin B₁₂ and/or folic acid; and to treat hemolytic anemia, such as, for example, adrenocortical steroids, e.g., corticoids. See e.g., Resegotti et al., Panminerva Medica, 23:243-248 (1981); Kurtz, FEBS Letters, 14a:105-108 (1982); McGonigle et al., Kidney Int., 25:437-444 (1984); and Pavlovic-Kantera, Expt. Hematol., 8(supp. 8) 283-291 (1980), the contents of each of which are hereby incorporated by reference in their entireties.

Compounds that enhance the effects of or synergize with erythropoietin are also useful as adjuvants herein, and include but are not limited to, adrenergic agonists, thyroid hormones, androgens, hepatic erythropoietic factors, erythrotropins, and erythrogenins, See for e.g., Dunn, “Current Concepts in Erythropoiesis”, John Wiley and Sons (Chichester, England, 1983); Kalmani, Kidney Int., 22:383-391 (1982); Shahidi, New Eng. J. Med., 289:72-80 (1973); Urabe et al., J. Exp. Med., 149:1314-1325 (1979); Billat et al., Expt. Hematol., 10:133-140 (1982); Naughton et al., Acta Haemat, 69:171-179 (1983); Cognote et al. in abstract 364, Proceedings 7th Intl. Cong. of Endocrinology (Quebec City, Quebec, Jul. 1-7, 1984); and Rothman et al., 1982, J. Surg. Oncol., 20:105-108 (1982). Methods for stimulating hematopoiesis comprise administering a hematopoietically effective amount (i.e., an amount which effects the formation of blood cells) of a pharmaceutical composition containing polynucleotides and/or polypeptides of the invention (and/or agonists or antagonists thereof) to a patient. The polynucleotides and/or polypeptides of the invention and/or agonists or antagonists thereof is administered to the patient by any suitable technique, including but not limited to, parenteral, sublingual, topical, intrapulmonary and intranasal, and those techniques further discussed herein. The pharmaceutical composition optionally contains one or more members of the group consisting of erythropoietin, testosterone, progenitor cell stimulators, insulin-like growth factor, prostaglandins, serotonin, cyclic AMP, prolactin, triiodothyzonine, methenolene, stanozolol, and nandrolone, iron preparations, vitamin B₁₂, folic acid and/or adrenocortical steroids.

In an additional embodiment, the antibody and antibody compositions of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, LEUKINE™ (SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the antibody and antibody compositions of the invention are administered in combination with fibroblast growth factors. Fibroblast growth factors that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

Additionally, the antibody and antibody compositions of the invention may be administered alone or in combination with other therapeutic regimens, including but not limited to, radiation therapy. Such combinatorial therapy may be administered sequentially and/or concomitantly.

Kits

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In an alternative embodiment, a kit comprises an antibody fragment that immunospecifically binds to B Lymphocyte Stimulator. In a specific embodiment, the kits of the present invention contain a substantially isolated B Lymphocyte Stimulator polypeptide as a control. Preferably, the kits of the present invention further comprise a control antibody which does not react with B Lymphocyte Stimulator. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to B Lymphocyte Stimulator (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized B Lymphocyte Stimulator. The B Lymphocyte Stimulator provided in the kit may also be attached to a solid support. In a more specific embodiment the detecting means of the above-described kit includes a solid support to which B Lymphocyte Stimulator is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to B Lymphocyte Stimulator can be detected by binding of the said reporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with B Lymphocyte Stimulator, and means for detecting the binding of B Lymphocyte Stimulator to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound B Lymphocyte Stimulator obtained by the methods of the present invention. After B Lymphocyte Stimulator binds to a specific antibody, the unbound serum components are removed by washing, reporter-labeled anti-human antibody is added, unbound anti-human antibody is removed by washing, and a reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-B Lymphocyte Stimulator antibody on the solid support. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate.

The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant B Lymphocyte Stimulator, and a reporter-labeled anti-human antibody for detecting surface-bound anti-B Lymphocyte Stimulator antibody.

In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908.

In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1563 to 1880.

In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1881 to 2128.

In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1 to 1562.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1881 to 2128, and in which said antibody or fragment thereof immunospecifically binds to the membrane-bound form of B Lymphocyte Stimulator.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1563 to 1880, and in which said antibody or fragment thereof immunospecifically binds to the soluble form of B Lymphocyte Stimulator.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1881 to 2128, and in which said antibody or fragment thereof immunospecifically binds to the membrane-bound form of B Lymphocyte Stimulator.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1563 to 1880, and in which said antibody or fragment thereof immunospecifically binds to the soluble form of B Lymphocyte Stimulator.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128. and in which said VL and said VH domains are derived from the same scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising an amino acid sequence of one of SEQ ID NOS: 2129 to 3227 wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.

In specific embodiments, the antibody or fragment thereof of the invention is a whole immunoglobulin molecule.

In specific embodiments, the antibody or fragment thereof of the invention is a Fab fragment.

In specific embodiments, the antibody or fragment thereof of the invention is a Fv fragment.

In specific embodiments, the present invention encompasses a chimeric protein comprising the antibody or fragment thereof of the invention covalently linked to a heterologous polypeptide.

In specific embodiments, the present invention encompasses a composition comprising two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VH domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention encompasses a composition comprising two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VL domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention encompasses a composition comprising two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VL domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and wherein each type of antibody or fragment thereof further comprises a VH domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention encompasses a composition comprising two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VH CDR3 having an amino acid sequence of one of SEQ ID NOS: 3129 to 3227.

In specific embodiments, the present invention encompasses a panel of two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VH domain from a different scFv having an amino acid sequence of one of SEQ ID NO: 1 to 2128.

In specific embodiments, the present invention encompasses a panel of two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VL domain from a different scFv having an amino acid sequence of one of SEQ ID NO: 1 to 2128.

In specific embodiments, the present invention encompasses a panel of two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VL domain from a different scFv having an amino acid sequence of one of SEQ ID NO: 1 to 2128 and wherein each type of antibody or fragment further comprises a VH domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention encompasses a panel of two or more antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VHCDR3 from a different scFv having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227.

In specific embodiments, the antibodies or fragments thereof of the antibody panel of the invention, are each in a well of a 96 well plate.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1881 to 1908, wherein the antibody of fragment thereof immunospecifically binds the membrane-bound form of B Lymphocyte Stimulator.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1563 to 1569, wherein said antibody of fragment thereof immunospecifically binds the soluble form of B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1881 to 2128, wherein the antibody of fragment thereof immunospecifically binds the membrane-bound form of B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1563 to 1880, wherein said antibody of fragment thereof immunospecifically binds the soluble form of B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and in which said VL domain and said VH domain are derived from the same scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.

In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VHCDR3 from an scFv having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.

In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VH domain encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VL domain encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VH domain encoded by a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence encoding a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VL domain encoded by a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence encoding a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a CDR encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a CDR from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a CDR encoded by a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence encoding a CDR from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VH CDR3 encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a VH CDR3 having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227.

In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VH CDR3 encoded by a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence encoding a VH CDR3 having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227.

In specific embodiments, the present invention provides a method for detecting of aberrant expression of B Lymphocyte Stimulator, comprising:

assaying the level of B Lymphocyte Stimulator expression in cells or a tissue sample of an individual using one or more antibodies or fragments or variants thereof that immunospecifically bind B Lymphocyte Stimulator; and

comparing the level of B Lymphocyte Stimulator assayed in the cells or a tissue sample with a standard level of B Lymphocyte Stimulator or a level of B Lymphocyte Stimulator in cells or a tissue sample from an individual without aberrant B Lymphocyte Stimulator expression, wherein an increase or decrease in the assayed level of B Lymphocyte Stimulator or level in cells or a tissue sample from an individual without aberrant B Lymphocyte Stimulator expression compared to the standard level of B Lymphocyte Stimulator is indicative of aberrant expression.

In specific embodiments, the present invention provides a method for diagnosing a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising:

administering to a subject an effective amount of a labeled antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator;

waiting for a time interval following the administering for permitting the labeled antibody or fragment thereof to preferentially concentrate at sites in the subject where B Lymphocyte Stimulator is expressed;

determining background level; and

detecting the labeled antibody or fragment thereof in the subject, such that detection of labeled antibody or fragment thereof above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of B Lymphocyte Stimulator.

In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above comprises a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.

In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above comprises a VH CDR3 having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227.

In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent.

In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent wherein the diagnostic agent is horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.

In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent wherein the diagnostic agent is fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.

In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent wherein the diagnostic agent is ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y or ⁹⁹Tc.

In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent wherein the diagnostic agent is luciferase, luciferin or aequorin.

A pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier.

A pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier.

A pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and a pharmaceutically acceptable carrier.

A pharmaceutical composition comprising at least one antibody or fragment thereof of comprising an amino acid sequence of one of SEQ ID NOS: 2129 to 3227 wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier.

A method of treating, preventing or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising administering to an animal in need thereof the pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and a pharmaceutically acceptable carrier in an amount effective to treat, prevent or ameliorate the disease or disorder. This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.

A method of treating, preventing or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising administering to an animal in need thereof the pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier in an amount effective to treat, prevent or ameliorate the disease or disorder. This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.

A method of treating, preventing or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising administering to an animal in need thereof the pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and a pharmaceutically acceptable carrier in an amount effective to treat, prevent or ameliorate the disease or disorder. This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.

A method of treating, preventing or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising administering to an animal in need thereof the pharmaceutical composition of comprising at least one antibody or fragment thereof of comprising an amino acid sequence of one of SEQ ID NOS: 2129 to 3227 wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier in an amount effective to treat, prevent or ameliorate the disease or disorder. This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.

This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.

EXAMPLES Abbreviations

0.2 M Tris-HCl, 0.5 mM EDTA, 0.5 M sucrose (TES) 1-ethyl-3-[3-dimethylaminopropyl]carbo diimide hydrochloride (EDC) 2TY supplemented with 100 μg/ml ampicillin and 2% glucose (2TYAG) 2TY supplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin (2TYAK)

3,3′,5,5′-Tetramethyl Benzidine (TMB)

50% inhibitory concentration (IC₅₀) 6×PBS containing 18% Marvel blocking solution (6×MPBS)

Absorbance (A)

Bovine serum albumin (BSA) Enzyme linked immunosorbent assay (ELISA) Foetal calf serum (FCS) Heavy chain variable (V_(H)) Hepes buffered saline (HBS) Horseradish peroxidase (HRP)

Immobilised Metal Affinity Chromatography (IMAC) Isopropyl β-D-thiogalactopyranoside (IPTG)

Light chain variable (V_(L)) Multiplicity of infection (MOI) N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (Hepes)

Nanomolar (nM) N-Hydroxysuccinimide (NHS)

PBS containing 3% Marvel (MPBS)

Phosphate Buffered Saline (PBS)

Phosphate Buffered Saline+0.1% (v/v) Tween 20 (PBST)

Picomolar (pM)

Single chain fragment variable (scFv)

Tumour Necrosis Factor-alpha (TNF-α) Tumour Necrosis Factor-beta (TNF-(β)

TNF-related apoptosis inducing ligand (TRAIL)

DEFINITIONS

In the following section “immobilized B Lymphocyte Stimulator” refers to a soluble form of B Lymphocyte Stimulator or biotinylated B Lymphocyte Stimulator coated on a plastic assay plate (e.g., a 96 well plate), but does not refer to histidine tagged B Lymphocyte Stimulator coated on a plastic assay plate; “biotinylated B Lymphocyte Stimulator” is a soluble form of B Lymphocyte Stimulator except when used to coat an ELISA plate, in which case it would be “immobilized B Lymphocyte Stimulator.” Membrane bound forms of B Lymphocyte Stimulator include, but are not limited to, U937 and P388 plasma membranes. Antibodies of the present invention are defined as able to bind the membrane bound and/or soluble forms of B Lymphocyte Stimulator according to the assays described in Examples 1 through 19.

Example 1 Antibodies Immunospecifically Binding to Soluble and Membrane-Bound B Lymphocyte Stimulator

A library of phage was screened in an assay to identify those phage displaying scFvs that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator. Phage displaying scFvs that bound to immobilized B Lymphocyte Stimulator were identified after panning on immobilized B Lymphocyte Stimulator and assessment by ELISA for binding to immobilized B Lymphocyte Stimulator. The B Lymphocyte Stimulator that was immobilized on plates for these assays was purified from supernatants of Sf9 cells infected with a baculovirus expression construct as described in Moore et al., Science 285:260-263 which is hereby incorporated by reference in its entirety. Each of the identified scFvs were then sequenced. Certain sequences were isolated multiple times, thus a panel (panel 1) containing one member of each unique sequences was generated and further characterized for their ability to immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator.

The derived amino acid sequences of these scFvs are shown in Table 1 above. The individual V_(H) and V_(L) segments of the scFvs were aligned to the known human germline sequences in V-BASE (Tomlinson et al, which can accessed on the United Kingdom Medical Research Council (MRC) Centre for Protein Engineering website) and the closest germline identified.

Example 2 Specificity of scFvs for B Lymphocyte Stimulator and Membrane-Bound B Lymphocyte Stimulator

The specificity of each of the scFvs for both B Lymphocyte Stimulator and membrane-bound B Lymphocyte Stimulator was determined by phage ELISA. B Lymphocyte Stimulator was immobilised onto plastic as a purified soluble form of the protein or as a membrane-bound form present on plasma membrane preparations from the human macrophage-like cell line, U937.

Maintenance of U937 Cells

U937 cells are a human monocyte-like, histiocytic lymphoma cell line known to express B Lymphocyte Stimulator on their plasma membranes. They were maintained in RPMI-1640 supplemented with 4 mM L-glutamine, 10% FCS, 10 U penicillin, 100 g/ml streptomycin (all reagents from Sigma). The cells were thawed from frozen stock and are either used for plasma membrane preparation, or split 1:5, after 2 days in culture when the cell density reaches 1×10⁶/ml.

Preparation of U937 Plasma Membranes

To prepare plasma membranes, 1×10⁹ U937 cells were harvested from their culture medium by centrifugation at 1000 rpm at 4° C. for 5 minutes in a benchtop centrifuge. The cells were resuspended in 40 ml 12 mM Tris, pH 7.5, 250 mM sucrose and placed on ice. The cells are then lysed using a hand-held electric homogenizer (Labortechnik IKA Ultra-Turrax) for four, one minute, bursts. To check that cell lysis had occurred, 10 μl cell lysate was added to 10 μl Trypan blue and the cell lysate was examined under a microscope. After confirming lysis, the homogenate was centrifuged at 270×g, for 10 minutes at 4° C. to pellet the nuclear fraction and the supernatant was retained. The supernatant was centrifuged at 8000×g, 10 mins, 4° C., to pellet the mitochondrial and lysosomal fractions and the supernatant was retained. The supernatant was then centrifuged at 100000×g, 60 mins, 4° C. to pellet the plasma membrane enriched fraction. The supernatant was discarded and the plasma membrane pellet was resuspended in 1 ml PBS and stored at −70° C. The protein concentration of the plasma membrane fraction was determined using a protein quantification kit (Biorad). Typical yields were between 5 and 10 mg of plasma membranes.

Phage ELISA

To determine the specificity of each of the unique scFvs, a phage ELISA was performed for each scFv against human B Lymphocyte Stimulator, U937 plasma membranes, TNFα (R&D Systems, Minneapolis, Minn.), BSA and uncoated well. Individual E. coli colonies containing a phagemid representing one of the unique scFvs from panel 1 were inoculated into 96-well plates containing 100 μl 2TYAG medium per well. Plates were incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage was added to each well to a MOI of 10 and the plates were incubated for a further 1 hour at 37° C. The plates were centrifuged in a benchtop centrifuge at 2000 rpm for 10 minutes. The supernatant was removed and cell pellets were resuspended in 100 μl 2TYAK and incubated at 30° C. overnight, shaking. The next day, plates were centrifuged at 2000 rpm for 10 min and the 100 ml phage-containing supernatant from each well carefully transferred into a fresh 96-well plate. Twenty μl of 6×MPBS was added to each well, and incubated at room temperature for 1 hour to pre-block the phage prior to ELISA.

Flexible 96-well plates (Falcon) were coated overnight at 4° C. with human B Lymphocyte Stimulator (1 μg/ml) in PBS, U937 plasma membranes (10 μg/ml) in PBS, TNFα (1 μg/ml) in PBS, BSA (1 μg/ml) in PBS, or PBS. After coating, the solutions were removed from the wells, and the plates were blocked for 1 hour at room temperature in MPBS. The plates were washed 3 times with PBS and then 50 μl of pre-blocked phage was added to each well. The plates were incubated at room temperature for 1 hour and then washed with 3 changes of PBST followed by 3 changes of PBS. To each well, 50 μl of an anti-gene VIII-HRP conjugate (Pharmacia) at a 1 to 5000 dilution in MPBS was added and the plates incubated at room temperature for 1 hour. Each plate was washed three times with PBST followed by three times with PBS. Then 50 ml of an HRP-labelled anti-mouse polymer (DAKO EnVision) diluted 1/50 in 3% MPBS was added and incubated for 1 hour at room temperature. Each plate was then washed three times with PBST followed by three times with PBS. Fifty μl of TMB substrate was then added to each well, and incubated at room temperature for 30 minutes or until colour development. The reaction was stopped by the addition of 25 μl of 0.5 M H₂SO₄. The signal generated was measured by reading the absorbance at 450 nm (A₄₅₀) using a microtiter plate reader (Bio-Rad 3550).

The results for 3 clones (I006E07, 1008D05 and I016F04) are shown in FIG. 1. All 3 scFvs recognize immobilized B Lymphocyte Stimulator and U937 plasma membranes but do not recognize TNFα, BSA or an uncoated well (PBS only). These results indicate that these scFvs specifically recognize immobilized B Lymphocyte Stimulator and membrane-bound B Lymphocyte Stimulator.

Example 3 Inhibition in an In Vitro Receptor Binding Assay by Phage ScFvs

All of the unique phage scFvs in panel 1 were assessed for their ability to inhibit soluble B Lymphocyte Stimulator binding to its cognate receptor on IM9 cells.

Biotinylation of B Lymphocyte Stimulator

One hundred μg of either human or mouse B Lymphocyte Stimulator was dialysed overnight at 4° C. against 50 mM sodium bicarbonate (sodium hydrogen carbonate) pH8.5 using a slide-a-lyzer cassette (Pierce). The next day, NHS-biotin (Pierce) was dissolved in DMSO to 13.3 mg/ml. This was then added to the B Lymphocyte Stimulator at a molar ratio of 20:1 biotin:B Lymphocyte Stimulator, mixed and incubated on ice for 2 hours. The biotinylated B Lymphocyte Stimulator was then dialysed back into sterile PBS (Sigma) using a slide-a-lyzer cassette overnight at 4° C. The biological activity of the biotinylated B Lymphocyte Stimulator was confirmed using the receptor binding inhibition assay (see below).

Maintenance of IM9 Cells

IM9 cells are a human B lymphocyte cell line. They were maintained in RPMI-1640 supplemented with 4 mM L-glutamine, 10% FCS, 10 U penicillin, 100 g/ml streptomycin (all reagents from Sigma). The cells are thawed from frozen stock and can be used in assays after 5 days in culture when they reach a density of 4-8×10⁵/ml.

Receptor Binding Inhibition Assay

Individual E. coli colonies containing a phagemid representing one of the unique scFvs from panel 1 were inoculated into 96-well plates containing 100 μl 2TYAG medium per well. Plates were incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage was added to each well to a MOI of 10 and the plates were incubated for a further 1 hour at 37° C. The plates were centrifuged in a benchtop centrifuge at 2000 rpm for 10 minutes. The supernatant was removed and cell pellets were resuspended in 100 μl 2TYAK and incubated at 30° C. overnight, shaking. The next day, plates were centrifuged at 2000 rpm for 10 min and the 100 μl phage-containing supernatant from each well carefully transferred into a fresh 96-well plate. Phage were diluted 1 in 2 in MPBS prior to use.

Flat-bottomed 96-well plates (Costar) were coated with 100 ml per well of a 1:10 dilution of poly-L-lysine (Sigma) in PBS for 1 hour at room temperature. The plates were then washed twice with water, allowed to air-dry and placed at 4° C. overnight. One hundred μl of IM9 cells (at 10⁶/ml in RPMI-1640 culture medium) were then added to each well. Plates were then centrifuged at 3200 rpm for 5 mins to pellet the cells. The media was carefully aspirated and 200 μl of MPBS added to each well. The plates were then allowed to block for 1 hour at room temperature.

To a separate 96-well plate 10 ml of biotinylated B Lymphocyte Stimulator (at 162.5 ng/ml) in MPBS was added to each well to give a final concentration of 25 ng/ml. Fifty-five μl of each appropriate phage supernatant was added to each well and the final volume in each well was 65 μl. Plates were then incubated at room temperature for 30 minutes.

The IM9 coated plates were washed twice in PBS, tapped dry and immediately 50 μl of the phage/biotinylated-B Lymphocyte Stimulator mix was added and incubated at room temperature for 1 hour. Plates were washed three times in PBST and three times in PBS, tapped dry and 50 μl of streptavidin-Delfia (Wallac) was added to each well at 1:1000 dilution in the Manufacturer's assay buffer. The plates were then incubated at room temperature for 1 hour and washed six times in Delfia wash solution (Wallac). After tapping the plates dry, 100 μl per well of Delfia enhancement solution (Wallac) was added. The plates were gently tapped to encourage micelle formation, incubated at room temperature for 10 minutes, and fluorescence read on a Wallac 1420 workstation at 6520 nM.

Results for 3 phage scFvs (I001C09, I018D07 and I016H07) that inhibited the binding of biotinylated B Lymphocyte Stimulator are shown in FIG. 2. Maximal binding of biotinylated B Lymphocyte Stimulator to its receptor (bio-B Lymphocyte Stimulator only), the background signal in the absence of biotinylated B Lymphocyte Stimulator (no bio-B Lymphocyte Stimulator), and results with an irrelevant (i.e., does not recognize B Lymphocyte Stimulator) phage antibody are also shown. All 3 phage scFvs inhibited biotinylated B Lymphocyte Stimulator binding to its receptor on IM9 cells, identifying these scFvs as scFvs that bind the soluble form of B Lymphocyte Stimulator. These scFvs also bind to U937 membranes, thus they also bind the membrane bound form of B Lymphocyte Stimulator.

Forty-eight of the scFvs from panel 1 that demonstrated the greatest inhibition as phage particles in this assay were chosen for further study. These 48 scFvs are listed in Table 3.

TABLE 3 scFvs that Inhibit the Binding of Biotinylated-B Lymphocyte Stimulator to its Receptor Antibody Antibody Antibody Antibody Antibody I008C02 I029D07 I008C03 I008C12 I028A06 I022E02 I061E07 I007H08 1061H01 I031C03 I018C02 I006D07 I008A11 I006D08 I031F02 I008B01 I017D10 I061D02 I026E03 I031F09 I016F04 I007B03 I008A09 I027A07 I031G11 I016E05 I018C10 I007F11 I016H07 I050A07 1018H08 I001C09 I037E07 I021B05 I050A12 I018H09 I018D07 I037E12 I031G10 I050B11 I029F11 I016F02 I031G08 I051C04 I022D01 I031C07 I003F12 I012A06

Example 4 Specificity of Anti-B Lymphocyte Stimulator Antibodies

The specificity of the 48 scFvs listed in Table 3 for human and murine B Lymphocyte Stimulator was determined using phage ELISA.

Phage ELISA

To determine the specificity of the 48 scFvs, a phage ELISA was performed against human and mouse B Lymphocyte Stimulator, and a panel of related and unrelated human antigens: Fas ligand, TRAIL, TNFα, TNFβ, and PBS. The Fas ligand, TRAIL, TNFα, and TNFβ antigens were obtained from R&D Systems, Minneapolis, Minn. Individual E. coli colonies containing phagemid were inoculated into 5 ml 2YTAG and incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage (Pharmacia) was added to each tube to a MOI of 10 and incubated for 30 minutes at 37° C. for 1 hour, the first 30 minutes static and the final 30 minutes with gentle shaking. Cells were pelleted by centrifugation at 3,500 rpm for 10 minutes and the supernatant discarded. Cell pellets were resuspended in 5 ml 2TYAK and incubated at 30° C. overnight with shaking. The next day, the cells were pelleted by centrifugation at 3,500 rpm for 10 minutes. The phage-containing supernatant (5 ml) was carefully transferred to a fresh tube, 1 ml of 6 MPBS was added, and the tube was incubated at room temperature for 1 hour to pre-block the phage prior to ELISA.

All antigens were coated at 1 μg/ml. ELISAs were performed essentially as described in Example 2. The only exception to this being the detection of phage antibody binding to mouse B Lymphocyte Stimulator where the step involving incubation with the HRP-labelled anti-mouse polymer was omitted. Binding to mouse B Lymphocyte Stimulator was detected with TMB as in Example 2.

All 48 scFvs are specific for immobilized human B Lymphocyte Stimulator and 43 out of the 48 scFvs cross-react with immobilized mouse B Lymphocyte Stimulator but not with any other unrelated or related antigen tested. I008C03, I007F11, I037E07, I037E12, and I016H07 did not bind murine B Lymphocyte Stimulator. Results for two scFvs, I022D01 and I031F02, are shown in FIG. 3. Both these scFvs specifically recognize human and mouse B Lymphocyte Stimulator but not any other unrelated or related antigen tested.

Example 5 Specificity for the Membrane-Bound Form of B Lymphocyte Stimulator

The specificity of 48 scFvs for membrane-bound B Lymphocyte Stimulator was determined by the phage ELISA described in Example 2. B Lymphocyte Stimulator was immobilised onto plastic as a membrane-bound form present on plasma membranes preparations from the human macrophage-like cell line, U937. This cell line is known to express the membrane-bound form of human B Lymphocyte Stimulator.

To demonstrate that this binding is specific for membrane-bound B Lymphocyte Stimulator, a competition ELISA was developed to determine if the ELISA signal for an individual antibody on U937's could be competed out by pre-incubation with either B Lymphocyte Stimulator or TNFα. An anti-B Lymphocyte Stimulator antibody that also recognizes membrane-bound B Lymphocyte Stimulator would be expected to demonstrate a signal reduction with free B Lymphocyte Stimulator but not free TNFα.

Competition ELISA

Individual E. coli colonies containing phagemid for each of the 48 scFvs listed in Table 3 were inoculated into 5 ml 2YTAG and incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage (Pharmacia) was added to each tube to a MOI of 10 and incubated for 30 minutes at 37° C. for 1 hour, the first 30 minutes static and the final 30 minutes with gentle shaking. Cells were pelleted by centrifugation at 3,500 rpm for 10 minutes and the supernatant discarded. Cell pellets were resuspended in 5 ml 2TYAK and incubated at 30° C. overnight with shaking. The next day, the cells were pelleted by centrifugation at 3,500 rpm for 10 minutes. The phage-containing supernatants (5 ml) were carefully transferred to a fresh tube.

For each of the 48 scFvs listed in Table 3, two aliquots of 20 μl 6×MPBS were pipetted into separate wells of a 96-well plate (Greiner). The first aliquot was supplemented with B Lymphocyte Stimulator to a final concentration of 0.5 μg/ml. The second aliquot was supplemented with TNF-α to a final concentration of 0.5 μg/ml. Each experiment was performed in triplicate. One hundred μl of each phage supernatant was then added to each aliquot and mixed by pipetting up and down. The phage were incubated (±competing antigen) at room temperature for 1 hour.

Flexible 96-well plates (Falcon) were coated overnight at 4° C. with 50 μl of 10 μg/ml U937 plasma membranes. After coating, the plates were washed 3 times with PBS and blocked for 1 hour at room temperature with 200 μl MPBS. The plates were washed 3 times with PBS and 50 μl of phage (±competing antigen) was added to each appropriate well. The plates were incubated at room temperature for 1 hour and then washed with 3 changes of PBST followed by 3 changes of PBS. To each well, 50 μl of a mouse anti-gene VIII-HRP conjugate (Pharmacia) at a 1:5000 dilution in MPBS was added and the plates incubated at room temperature for 1 hour. Each plate was washed three times with PBST followed by three times with PBS. Then 50 ml of an HRP-labelled anti-mouse polymer (DAKO EnVision) diluted 1:50 in 3% MPBS was added and incubated for 1 hour at room temperature. Each plate was then washed three times with PBST followed by three times with PBS. Fifty μl of TMB substrate was then added to each well, and incubated at room temperature for 30 to 60 minutes or until color development. The reaction was stopped by the addition of 25 μl of 0.5 M H₂SO₄. The signal generated was measured by reading the absorbance at 450 nm (A₄₅₀) using a microtiter plate reader (Bio-Rad 3550).

All 48 scFvs bind to U937 plasma membrane preparations. This signal could be competed out by pre-incubation of the phage antibody with B Lymphocyte Stimulator but not by pre-incubation with TNF-α. This indicates that the 48 scFvs specifically recognize membrane-bound B Lymphocyte Stimulator as well as soluble B Lymphocyte Stimulator. Typical results are exemplified by scFvs I031F09, I050A12 and I051C04 and are shown in FIG. 4. All 3 scFvs demonstrate binding to U937 plasma membranes. This binding was specifically competed out with B Lymphocyte Stimulator but did not compete with TNF-α, demonstrating specific recognition of membrane-bound B Lymphocyte Stimulator.

Example 6 scFv Off-Rate Determinations

All off-rate determinations were performed on BIAcore 2000 machines, using the BIAcore 2000 Control Software and evaluated using the BIAevaluation 3.0 software.

Preparation of a Low Density B Lymphocyte Stimulator Surface

A 500RU surface was prepared for kinetic studies with purified scFvs. A low density B Lymphocyte Stimulator surface (500 RU B Lymphocyte Stimulator coupled) was prepared in flow cell 2 by amine coupling to a CM5 chip. A new CM5 chip was inserted into the BIAcore and a sensorgram initiated with HBS buffer at a flow rate of 5 μl/min. The NHS and EDC coupling solutions (BIAcore) were mixed according to manufacturer's instructions and 30 μl injected over the CM5 surface. Fifty μl of B Lymphocyte Stimulator at 1 μg/ml in 10 mM sodium acetate buffer, pH4, was then injected followed by 30 μl of ethanolamine-HCl solution (BIAcore). The flow rate was then adjusted to 20 μl/min and 10 μl of 4M guanidine hydrochloride in HBS injected over the surface. This strips the surface of non-covalently bound B Lymphocyte Stimulator.

Measurement of scFv Off-Rate Kinetics on the Low Density Surfaces

The chip containing the low density B Lymphocyte Stimulator surface was inserted in to the BIAcore. A dilution series of purified scFvs was prepared in HBS, typically 50 μg/ml doubling dilutions down to 1.5 ug/ml. The dilution series was then injected sequentially over the low density B Lymphocyte Stimulator surface (and blank control) using the following program:

MAIN FLOWCELL 1,2,3,4 APROG genab r1d1 ab1 APROG genab r1d2 ab2 APROG genab r1d3 ab3 APROG genab r1d4 ab4 APROG genab r1d5 ab5 APROG genab r1d6 ab6 APPEND CONTINUE END DEFINE APROG genab PARAM %Abpos %AbId FLOW 20 KINJECT %Abpos 200 80 INJECT r1c6 10!guanidine hydrochloride regeneration step EXTRACLEAN END

Bound scFvs were removed by injecting 10 μl 4M GuHCl in HBS over the surface between scFv samples.

The binding curves for individual scFvs were analyzed using the BIAevaluation software to determine antibody off-rates. Kinetic analysis for a typical scFv antibody, I003C02, is shown in FIG. 5. I003C02 has a K_(off)=6×10 ⁻³ s⁻¹.

Example 7 Inhibition in an In Vitro Receptor Binding Assay by scFv Antibodies

The 48 scFvs listed in Table 3 were purified and assessed for their ability to inhibit B Lymphocyte Stimulator binding to its receptor on IM9 cells.

Purification of scFv

To determine the inhibitory potency of anti-B Lymphocyte Stimulator scFv, scFv's were first prepared by IMAC. 2TYAG (5 ml) was inoculated with a single colony and grown overnight at 30° C., shaking. This overnight culture was then used to inoculate 500 ml of 2TY containing 100 μg/ml ampicillin and 0.1% Glucose, and grown at 30° C., shaking, until an A₆₀₀ of 1.0 was attained. IPTG was added to 1 mM and the culture was grown for a further 3.5 hours at 30° C.

Cells were harvested by centrifugation at 5,000 rpm, and resuspended in 10 ml of TES. A further 15 ml of a 1:5 dilution (in water) of TES was added, and the cell suspension incubated on a turning wheel at 4° C. for 30 minutes. This causes osmotic shock and yields a periplasmic extract containing the scFv. Residual cells and debris were pelleted by centrifugation at 9,000 rpm for 20 minutes at 4° C. The supernatant was transferred to a new tube, and 50 μl of 1 M MgCl₂ added. Two ml of a Ni-NTA agarose (Qiagen), pre-washed with buffer (50 mM sodium phosphate, pH 8, 300 mM NaCl) together with a protease inhibitor tablet (Boehringer Mannheim) were then added to the periplasmic extract. The preparation was incubated, rotating, overnight at 4° C. The Ni-NTA was pelleted by centrifugation at 2,000 rpm for 5 minutes, and the supernatant was aspirated. The agarose beads were washed 3 times with 50 ml wash buffer, centrifuging to collect the agarose in between each wash. Ten ml of wash buffer was added after the final wash, and the slurry was loaded on to a polyprep column (BioRad). Two ml elution buffer (50 mM NaPi (sodium phosphate), pH 8, 300 mM NaCl, 250 mM imidazole) was added to the drained agarose, and the eluate was collected. IMAC purified scFv was buffer exchanged in to PBS by use of a Nap 5 column (Pharmacia) according to the manufacturer's instructions. The A₂₈₀ was read and the protein concentration determined using a molar extinction coefficient of 1 mg/ml protein=A₂₈₀ 1.4. Purified scFv was stored in 500 μl aliquots at −70° C.

Receptor Binding Inhibition Assay

Flat-bottomed 96-well plates (Costar) were coated with 100 ml per well of a 1:10 dilution of poly-L-lysine (Sigma) in PBS for 1 hour at room temperature. The plates were then washed twice with water, allowed to air-dry and placed at 4° C. overnight. One hundred μl of IM9 cells (at 10⁶/ml in RPMI-1640) were then added to each well. Plates were then centrifuged at 3200 rpm for 5 mins to pellet the cells. The media was carefully aspirated and 200 ml of MPBS added to each well. The plates were then left to block for 1 hour at room temperature.

To a separate 96-well plate, titrate test scFvs in MPBS, in triplicate, over a concentration range from 10 μg/ml down to 0.001 μg/ml were added. The final volume of test scFv in each well was 55 μl. Competition with unlabelled B Lymphocyte Stimulator was also included in every assay as a control. Unlabelled B Lymphocyte Stimulator, in MPBS, was typically titrated in triplicate, over a concentration range from 1 μg/ml down to 0.001 μg/ml. 10 μl of biotinylated-B Lymphocyte Stimulator (at 162.5 ng/ml) in MPBS was added to each well to give a final concentration of 25 ng/ml. Plates were then incubated at room temperature for 30 minutes.

The IM9 coated plates was washed twice in PBS, tapped dry and immediately 50 μl of the scFv/biotinylated-B Lymphocyte Stimulator mix was added and incubated at room temperature for 1 hour. Plates were washed three times in PBST and three times in PBS, tapped dry and 50 μl per well added of streptavidin-Delfia (Wallac) at 1:1000 dilution in the Manufacturer's assay buffer. The plates were then incubated at room temperature for 1 hour and washed six times in Delfia wash solution (Wallac). After tapping the plates dry, 100 μl per well of Delfia enhancement solution (Wallac) was added. The plates were gently tapped to encourage micelle formation, incubated at room temperature for 10 minutes, and fluorescence read on a Wallac 1420 workstation at 6520 nM.

Typical titration curves for two scFv antibodies, I007F11 and I050A07, are shown in FIG. 6. Unlabelled B Lymphocyte Stimulator competed for binding to its receptor with an IC₅₀ value of 0.8 nM. The IC₅₀ values for I007F11 and I050A07 are 7.9 nM and 17.1 nM, respectively. The assay was performed in triplicate and standard error bars are shown. The 9 scFvs that demonstrated the greatest inhibition as scFv are listed in Table 4. This data also confirms that these 9 scFvs recognize the soluble form of B Lymphocyte Stimulator.

TABLE 4 9 ScFvs that demonstrated greatest potency in B Lymphocyte Stimulator Receptor Binding Inhibition Assay ScFv Antibody I017D10 I022D01 I008A11 I006D08 I031F02 I050A12 I050B11 1051C04 I003F12S

Example 8 Antibodies Recognizing a Soluble Form of B Lymphocyte Stimulator

A library of phage was screened in an assay to identify those phage displaying scFvs that immunospecifically bind to the soluble but not the membrane-bound forms of B Lymphocyte Stimulator.

A phage library was screened for the ability to bind to biotinylated B Lymphocyte Stimulator. The phage were exposed to biotinylated B Lymphocyte Stimulator, allowed an interval of time to bind the biotinylated B Lymphocyte Stimulator. Phage binding bio-B Lymphocyte Stimulator were then isolated by capture on streptavidin coated magnetic beads.

The phage identified in the screen above (capture of Bio-B Lymphocyte Stimulator from solution) were then screened by ELISA for their ability to bind immobilized B Lymphocyte Stimulator. The scFv expressed by phage that bound immobilized B Lymphocyte Stimulator were then cloned and sequenced. Again, several sequences were identified multiple times, thus a panel (panel 2) consisting of on example of each phage expressing a unique scFv was then characterized further.

The derived amino acid sequences of these scFvs are shown in Table 1 above. The individual V_(H) and V_(L) segments of the scFvs were aligned to the known human germline sequences in V-BASE (Tomlinson et al, which can accessed on the United Kingdom Medical Research Council (MRC) Centre for Protein Engineering website) and the closest germline identified.

Example 9 Specificity for Soluble B Lymphocyte Stimulator

The scFvs were isolated from a library of phage based on their ability to bind a soluble form of B Lymphocyte Stimulator. Briefly, phage were preincubated with biotinylated B Lymphocyte Stimulator in solution. Phage that bound to this biotinylated B Lymphocyte Stimulator were then isolated using streptavidin coated magnetic beads.

The specificity of each of the unique scFvs for B Lymphocyte Stimulator and for the membrane-bound form of B Lymphocyte Stimulator, was determined by phage ELISA. B Lymphocyte Stimulator was immobilised onto plastic as a purified soluble form of the protein or as a membrane-bound form present on plasma membrane preparations from the human macrophage-like cell line, U937. Maintenance of U937 cells and plasma membrane preparations were performed as detailed in Example 2.

Phage ELISA

To determine the specificity of each of the scFvs, a phage ELISA was performed for each antibody against human B Lymphocyte Stimulator, U937 plasma membranes, TNFα, BSA and an uncoated well. Antigen coating conditions were as described in Example 2, apart from human B Lymphocyte Stimulator. B Lymphocyte Stimulator was first biotinylated (as described in Example 3) and coated at 1 μg/ml onto streptavidin coated plates (Reacti-Bind, Pierce) for 30 mins at room temperature. The plates were then washed, blocked and the phage ELISA performed as detailed in Example 2.

The results for 3 clones (I074B 12, I075F12 and I075A02) that bind the soluble but not the membrane-bound form of B Lymphocyte Stimulator are shown in FIG. 7. As a control, a phage antibody that recognizes TNFα, is also shown in FIG. 7. There is a small non-specific background signal on the U937 plasma membranes that is evident with both the anti-B Lymphocyte Stimulator scFvs as well as the anti-TNFα control. All 3 anti-B Lymphocyte Stimulator scFvs recognize B Lymphocyte Stimulator but not U937 plasma membranes, TNFα, BSA or an uncoated well (PBS only). This indicates that the scFvs do not bind the membrane-bound form of B Lymphocyte Stimulator. Further, The fact that these scFvs were isolated on the basis of their ability to bind soluble biotinylated B Lymphocyte Stimulator indicates that they bind the soluble form of B Lymphocyte Stimulator. Further confirmation of these scFvs' specificity for B Lymphocyte Stimulator is provided in Example 10.

Example 10 Inhibition in an In Vitro Receptor Binding Assay by Phage scFvs

All of the unique phage scFvs from panel 2 were assessed for their ability to inhibit B Lymphocyte Stimulator binding to its cognate receptor on IM9 cells. The biotinylation of B Lymphocyte Stimulator, maintenance of IM9 cells and receptor binding inhibition assay were performed as described in Example 3.

Results for two phage scFvs, I0025B09 and I026C04 are shown in FIG. 8. Maximal binding of biotinylated B Lymphocyte Stimulator to its receptor (bio-B Lymphocyte Stimulator only), the background signal in the absence of biotinylated B Lymphocyte Stimulator (no bio-B Lymphocyte Stimulator), and results with an irrelevant (i.e. does not recognize B Lymphocyte Stimulator) phage antibody are also shown. Both phage scFvs inhibited biotinylated B Lymphocyte Stimulator binding to its receptor on IM9 cells. 33 of the unique scFvs from panel 2 were identified for further study. These 33 scFvs demonstrated the greatest inhibition as phage particles in this assay and are listed in Table 5.

TABLE 5 Identification of 33 phage scFvs to free B Lymphocyte Stimulator that demonstrate the most significant inhibition of biotinylated-B Lymphocyte Stimulator binding to its receptor Antibody Antibody Antibody Antibody I026C04 I074B12 I073F04 I065D04 I003C06 I075A02 I078D08 I068C08 I025B09 I068B08 I078D02 I068F03 I027B12 I068B04 I075G01 I069B07 I025B06 I068C06 I071B03 I030A10 I075F12 I072B09 I002A01R I065D08 I078H08 I002A01K I065F08 I064C04 I026C04R I067B10 I064C07 I026C04K I067F05

Example 11 Specificity of Anti-B Lymphocyte Stimulator scFvs

The specificity of the 33 scFvs (listed in Table 5) for immobilized human and murine B Lymphocyte Stimulator was determined using phage ELISA.

Phage ELISA

To determine the specificity of the 33 scFvs, a phage ELISA was performed as described in Example 4 against human and mouse B Lymphocyte Stimulator, and a panel of related human antigens: TRAIL, LIGHT, TNFα, TNFβ, and an uncoated well (PBS only).

Typical results for two scFvs, I067F05 and I078D02 are shown in FIG. 9. A control antibody that specifically recognizes TNFα is also shown. Both anti-B Lymphocyte Stimulator scFvs specifically recognize immobilized human and mouse B Lymphocyte Stimulator but not any other antigen tested.

All 33 scFvs are specific for human B Lymphocyte Stimulator. 14/33 cross-react with mouse B Lymphocyte Stimulator but not with any other unrelated or related antigen tested.

Example 12 scFv Off-Rate Determinations

Off-rate determinations, preparation of a low density B Lymphocyte Stimulator surface and kinetic measurements were as detailed in Example 6.

The binding curves for individual scFvs were analysed using the BIAevaluation software to determine antibody off-rates. Kinetic analysis for a typical scFv antibody, I002A01, is shown in FIG. 10. I002A01 has a K_(off)=9×10 ⁻⁴ s⁻¹.

Example 13 Inhibition in an In Vitro Receptor Binding Assay by scFv Antibodies

The 33 scFvs identified in Table 5 were prepared as purified scFvs and assessed for their ability to inhibit B Lymphocyte Stimulator binding to its receptor on IM9 cells. The scFvs were purified and analysed in the receptor binding inhibition assay as described in Example 6.1.8.

Typical titration curves for two scFvs, I0068C06 and I074B12, are shown in FIG. 11. Unlabelled B Lymphocyte Stimulator competed for binding to its receptor with an inhibitory constant 50 (IC₅₀) value of 0.66 nM. The IC₅₀ values for I0068CO₆ and I074B12 are 61 nM and 13 nM, respectively. The assay was performed in triplicate and standard error bars are shown. The 7 scFvs that demonstrated the greatest inhibition as scFv are listed in Table 6.

TABLE 6 Identification of 7 scFvs to free B Lymphocyte Stimulator that demonstrate the most significant inhibition of biotinylated-B Lymphocyte Stimulator binding to its receptor as purified scFv's. Antibody I002A01-R I002A01-K I026C04-R I026C04-K I068C06 I075F12 I067B10

Example 14 ScFvs Recognizing Membrane-Bound B Lymphocyte Stimulator

A library of phage was screened in an assay to identify those phage displaying scFvs that immunospecifically bind to the membrane-bound but not the soluble form of B Lymphocyte Stimulator.

As a starting point, a library of phage expressing scFv antibodies were panned on immobilized HIS-tagged B Lymphocyte Stimulator. Phage isolated by panning were then screened for the ability to bind to HIS-tagged B Lymphocyte Stimulator. HIS-tagged B Lymphocyte Stimulator was obtained by expressing amino acids 71-285 of SEQ ID NO:3228 using the pQE9 vector (Qiagen Inc., Valencia, Calif.) in E. coli and purifying the expressed protein. This phage clones identified by this screen were then sequenced. After sequencing, A panel (panel 3) of phage each expressing a unique scFv that bound HIS-tagged B Lymphocyte Stimulator was generated and further characterized.

The derived amino acid sequences of the unique scFvs from panel 3 are shown in Table 1 above. The individual V_(H) and V_(L) segments of the scFvs were aligned to the known human germline sequences in V-BASE (Tomlinson et al, which can accessed on the United Kingdom Medical Research Council (MRC) Centre for Protein Engineering website) and the closest germline identified.

Example 15 Recognition of Membrane-Bound B Lymphocyte Stimulator

The specificity of each of the unique scFvs for both the membrane-bound form of B Lymphocyte Stimulator as well as for the soluble form of B Lymphocyte Stimulator, was determined by phage ELISA.

B Lymphocyte Stimulator was immobilised onto plastic either directly as a purified soluble form of the protein or biotinylated and coated on a streptavidin plate as in Example 9. Binding to HIS-tagged B Lymphocyte Stimulator was used as a primary screen for scFv's that would bind the membrane-bound form of B Lymphocyte Stimulator (see below). The membrane-bound form of B Lymphocyte Stimulator was presented as plasma membranes preparations from the human macrophage-like cell line, U937 or the murine cell line P388.

Mouse monoclonal antibodies have been raised against His-tagged B Lymphocyte Stimulator according to standard procedures. Characterization of these mouse monoclonal antibodies revealed that they specifically recognized both His-tagged B Lymphocyte Stimulator and the membrane-bound form of B Lymphocyte Stimulator on U937 cells, but not soluble B Lymphocyte Stimulator. Therefore, specific recognition of His-tagged B Lymphocyte Stimulator was used as supporting evidence for the recognition of the membrane-bound form of B Lymphocyte Stimulator by phage and scFv antibodies.

Phage ELISA

To determine the specificity of each of the scFvs, a phage ELISA was performed for each antibody against His-tagged human B Lymphocyte Stimulator, U937 plasma membranes, TNFα, BSA and an uncoated well. Antigen coating conditions were as described in 2. apart from human B Lymphocyte Stimulator. B Lymphocyte Stimulator was first biotinylated (as described in Example 3) and coated at 1 μg/ml onto streptavidin coated plates (Reacti-Bind, Pierce) for 30 mins at room temperature. The plates were then washed, blocked and the phage ELISA performed as detailed in Example 2.

The results for 3 clones, I079C01, I081C10 and I082A02, and a control phage antibody that recognizes TNFα, are shown in FIG. 12. All 3 scFvs recognize U937 plasma membranes (U937) and His-tagged B Lymphocyte Stimulator (HIS-B Lymphocyte Stimulator) but not, biotinylated B Lymphocyte Stimulator (bio-B Lymphocyte Stimulator) or an uncoated well (PBS). This indicates that the scFvs recognize the membrane-bound form of B Lymphocyte Stimulator.

Example 16 Specificity for Membrane-Bound B Lymphocyte Stimulator

The specificity of the scFvs for only the membrane-bound form of B Lymphocyte Stimulator, and not for the soluble form, was confirmed using a competition ELISA. This assay assesses the ability of test phage scFvs to bind to the membrane-bound form of B Lymphocyte Stimulator on U937 plasma membranes in the presence of different forms of competing B Lymphocyte Stimulator. Competing B Lymphocyte Stimulator was either the His-tagged form of B Lymphocyte Stimulator or soluble B Lymphocyte Stimulator. ScFvs specific for the membrane-bound B Lymphocyte Stimulator would be expected to be competed out by pre-incubation with His-tagged B Lymphocyte Stimulator but not by pre-incubation with soluble B Lymphocyte Stimulator.

Maintenance of U937 cells and plasma membrane preparations were performed as detailed in Example 2.

Competition ELISA

U937 plasma membranes (50 μl per well) were coated at 10 μg/ml in PBS onto Falcon 96-well plates overnight at 4° C.

Individual E. coli colonies containing a phagemid representing one of the unique scFvs from the panel 3 were inoculated into 50 ml tubes (Falcon) containing 5 ml 2TYAG medium. Tubes were incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage was added to each tube to an MOI of 10 and the tubes were incubated for a further 1 hour at 37° C. The tubes were centrifuged in a benchtop centrifuge at 3500 rpm for 10 minutes. The supernatant was removed and cell pellets were resuspended in 5 ml 2TYAK and incubated at 30° C. overnight, shaking. The next day, tubes were centrifuged at 3500 rpm for 10 min and the phage-containing supernatant carefully transferred into a fresh tube.

For each test phage antibody, 3 aliquots of 20 μl 18% marvel/6×PBS were transferred into separate wells of a 96-well plate. The first aliquot was supplemented with His-tagged B Lymphocyte Stimulator to a final concentration of 60 μg/ml. The second aliquot was supplemented with soluble B Lymphocyte Stimulator to a final concentration of 60 μg/ml. The third aliquot was not supplemented with any competing antigen. One hundred μl of phage supernatant was then added to each aliquot and left to block at room temperature for 1 hour.

The antigen-coated plates were washed once with PBS before the addition of 200 μl/well 3% marvel/PBS. These plates were left to block at 37° C. for 1 hour and were then washed once with PBS. Duplicate samples of 50 μl pre-blocked phage (above) were added to the antigen-coated plates and left at room temperature for 1 hour. Plates were washed 3× with PBS/0.1% Tween 20, then 3× with PBS. Fifty μl/well mouse anti-M13 HRP (Pharmacia) at 1/5000 in 3% Marvel/PBS was added and left for 1 hour at room temperature. Plates were washed 3 times with PBS/0.1% Tween 20, then 3 times with PBS. Fifty Owen HRP-labelled anti-mouse Envision polymer (DAKO) at 1/50 in 3% marvel/PBS was added and left for 1 hour at RT. Plates were washed 3 times with PBS/0.1% Tween 20, then 3 times with PBS. Next, 50 μl/well of TMB (Sigma) was added and plates left to develop for 30 to 60 minutes. When sufficient color has developed, 25 μl/well 0.5M H₂SO₄ was added to stop the reaction. The plates were read at 450 nm on a microtiter plate reader (Bio-Rad 3550).

The results for 3 clones, I079B04, I079F08 and I080B01, and a control phage antibody that recognizes TNFα, are shown in FIG. 13. All 3 scFvs recognize U937 plasma membranes (U937). This binding is competed out to background levels (i.e. comparable to the signal observed with the anti-TNFα phage antibody) in the presence of His-tagged B Lymphocyte Stimulator (HIS-B Lymphocyte Stimulator) but not biotinylated B Lymphocyte Stimulator (bio-B Lymphocyte Stimulator). This confirms that the scFvs specifically recognize the membrane-bound form but not the soluble form of B Lymphocyte Stimulator.

Example 17 High Throughput BIAcore Screen to Identify High Affinity scFvs

This is a 96-well screen where the test samples (scFvs) are derived from 1 ml periplasmic extracts of individual antibody expressing clones. Potentially higher affinity scFvs are then identified principally as those giving a large number of total RU's bound to a HIS-B Lymphocyte Stimulator surface in BIAcore. This method of ranking does assume approximately equal yields of scFv from each clone. Since this is not always the case, some scFvs may also be identified that simply express high levels of scFv. These can be discriminated from those of higher affinity by further characterization of the scFvs (see Example 18).

Preparation of ScFv from 1 ml E. coli Cultures

Individual E. coli colonies containing a phagemid representing one of the unique scFvs from panel 3 were inoculated into 96-well plates containing 100 μl 2TYAG medium per well. Eight wells on each plate were reserved for positive and negative control samples. The plate was grown overnight at 30° C. with shaking at 120 rpm.

Next day, 1 ml of 2TYAG+345 mM sucrose was added to each well of an autoclaved 96 deep well plate (Beckman). Twenty μl of each overnight culture was resuspended and transferred to the appropriate well of the deep well plate. The plate was grown for approximately 3.5 hours at 30° C. with shaking at 250 rpm (or until the OD₆₀₀=0.6). Fifty μl of 1M IPTG was added to 5 ml 2TY and 10 μl of this was added to each well. The plate was grown overnight at 30° C. with shaking at 250 rpm.

Plates were kept at 4° C. for the remainder of the procedure. The overnight plate (above) was centrifuged at 3500 rpm for 10 minutes at 4° C. to pellet the cells. The supernatant was decanted and each pellet resuspended in 100 μl TES (0.2M Tris HCl pH8.0, 0.5 mM EDTA, 0.5M sucrose) and transferred to a fresh 96 well plate. This plate was incubated on ice for 30 minutes and then centrifuged for 10 minutes at 3500 rpm at 4° C. to pellet the cell debris. During centrifugation, 15 μl of freshly made protease inhibitors cocktail (Roche, 1 tablet dissolved in 1.5 ml water) was added to each well of a fresh 96 well plate. Supernatants from the centrifuged plate were then transferred to the plate containing the protease inhibitors. The plate was centrifuged at 3500 rpm for 10 minutes at 4° C. and the supernatant was transferred to a further 96-well plate. This step was repeated at least once more or until there was no sign of any cell debris following centrifugation. Finally, the plate was covered in foil to prevent evaporation of samples during the BIAcore run.

Generation of a High Density HIS-B Lymphocyte Stimulator Surface

All BIAcore analysis was performed on BIAcore 2000 machines, using the BIAcore 2000 control software and evaluated using the BIAevaluation 3.0 software. A high density His-tagged B Lymphocyte Stimulator surface (>1000 RU HIS-B Lymphocyte Stimulator coupled) was prepared in flow cell 2 by amine coupling to a CM5 chip. A new CM5 chip was inserted into the BIAcore and a sensorgram started over flow cell 2 with HBS buffer at a flow rate of 5 μl/min. The NHS and EDC solution were mixed 1:1 before injecting 30 μl over the CM5 surface. Fifty μl HIS-B Lymphocyte Stimulator (at 10 μg/ml in Sodium acetate buffer, pH4) was injected and allowed to couple to the surface. Thirty μl of ethanolamine-HCl solution was then injected to block free NHS esters. Prior to using the chip, 10 μl of 4M Guanidine hydrochloride in HBS was injected over the surface to strip the surface of non-covalently bound B Lymphocyte Stimulator. A blank surface (no HIS-B Lymphocyte Stimulator) was also prepared over flow cell 1 so that non-specific binding effects can be subtracted from the HIS-B Lymphocyte Stimulator binding curves.

Typically, a 5000 RU His-tagged B Lymphocyte Stimulator surface was generated in this way and used for 96-well analysis of scFvs isolated from the periplasm of E. coli.

BIAcore Analysis

The 96-well plate containing periplasmic scFvs was secured inside the BIAcore. Two ml of 4M Guanidine hydrochloride in HBS was placed in a rack inside the BIAcore for regeneration of the HIS-B Lymphocyte Stimulator surface between samples. The sensorgram was run over flow cells 1 and 2 at a flow rate of 200 minute. The following method was run:

MAIN FLOWCELL 1,2,3,4

LOOP cycle STEP

APROG inj % pos ENDLOOP APPEND CONTINUE END

DEFINE LOOP cycle

LPARAM % pos

r1a1 r1b1 r1c1 r1d1 r1e1 r1f1 etc (all wells listed until r1h12)

END DEFINE APROG inj PARAM % pos FLOW 20

KINJECT % pos 35 30 !scfv injection QUICKINJECT r2f3 10 !regeneration

EXTRACLEAN END

When the run had finished, the sensorgram data for flow cell 1 was subtracted from the data for flow cell 2 for each sample using the BIAevaluation software. The clones were compared with one another principally by overall RU change as the scFv dissociates from the surface. In addition a few scFvs were identified as having potentially slower off-rates. An example of the dissociation section of a typical sensorgram for 8 scFvs is shown in FIG. 14. An anti-TNFα antibody that does not recognize B Lymphocyte Stimulator was included as a control. Of the 8 scFvs exemplified, I079F06 was identified for further study due to the relatively high numbers of RU's bound to the surface.

ScFvs were identified principally if they demonstrated a RU change of over 1200, a few were also identified as having potentially slower than typical off-rates. A total of 28 clones were chosen on these criteria and are listed in Table 7.

TABLE 7 Identification of 28 antibodies to membrane- bound B Lymphocyte Stimulator that demonstrate the most significant RU changes by BIAcore Antibody Antibody I079C01 I084C04 I082H08 I080E05 I079E02 I083B12 I079B05 I082G01 I079F06 I082G02 I079F08 I082C03 I079F11 I082A05 I079B12 I082D07 I080B01 I082B08 I080G09 I084A01 I099D03 I084B02 I080D03 I080A08 I080A03 I084C11 I083G03 I080G07

Example 18 scFv Affinity Determinations

The affinity (K_(D)) of the 28 scFvs was determined using the BIAcore.

Low Density HIS-B Lymphocyte Stimulator Surface for Kinetic Studies

500RU surfaces were used for kinetic studies of purified scFv binding to HIS-B Lymphocyte Stimulator. The method to prepare these surfaces was identical to the method described in Example 17, only smaller volumes of HIS-B Lymphocyte Stimulator were injected.

Measurement of scFv Binding Kinetics

The chip containing the low density HIS-B Lymphocyte Stimulator surface was inserted into the BIAcore. A dilution series for each of the 28 purified scFvs (prepared as in Example 6) were diluted in HBS (typically starting with 50 μg/ml scFv and double diluting down to 1.5 μg/ml). The dilution series was then injected sequentially over the blank control (flow cell 1) and low density HIS-B Lymphocyte Stimulator surface (flow cell 2) using the following program:

MAIN FLOWCELL 1,2,3,4 APROG genab r1d1 ab1 APROG genab r1d2 ab2 APROG genab r1d3 ab3 APROG genab r1d4 ab4 APROG genab r1d5 ab5 APROG genab r1d6 ab6 APPEND CONTINUE END DEFINE APROG genab PARAM %Abpos %AbId FLOW 20 KINJECT %Abpos 200 80 INJECT r2f3 10 EXTRACLEAN END

Bound scFv were removed by injecting 10 μl of 4M Guanidine hydrochloride in HBS (location r2f3 in the above program) over the surface between samples. Binding curves for individual scFv were analysed using the BIAevaluation software to determine antibody on- and off-rates.

A typical example of the binding curves generated for the scFv antibody I082C03 is shown in FIG. 15. The off-rate for this clone was calculated as 2×10⁻³ s⁻¹. The affinity of I082C03 was calculated as 20 nM, assuming 100% activity of the scFv. The 5 scFvs with the highest affinities as scFvs are given in Table 8.

TABLE 8 Identification of 5 antibodies to membrane-bound B Lymphocyte Stimulator that have the highest affinities as scFvs Affinity Antibody (K_(D)) I079F11 5 nM I079E02 10 nM  I082G02 6 nM I082H08 1 nM I099D03 4 nM

Example 19 Recognition of Mouse Membrane-Bound B Lymphocyte Stimulator

The ability of the 5 scFvs listed in Table 8 to also recognize murine membrane-bound B Lymphocyte Stimulator was determined using a competition ELISA. This assay assesses the ability of test phage scFvs to bind to the membrane-bound form of B Lymphocyte Stimulator on the murine cell line, P388, plasma membranes in the presence of different forms of competing human B Lymphocyte Stimulator. Competing B Lymphocyte Stimulator was either presented as the His-tagged form of B Lymphocyte Stimulator, or soluble B Lymphocyte Stimulator. ScFvs that recognize mouse membrane-bound B Lymphocyte Stimulator would give an ELISA signal on the P388 plasma membranes that is competed out by pre-incubation with HIS-tagged B Lymphocyte Stimulator but not by pre-incubation with soluble B Lymphocyte Stimulator.

Maintenance of P388.D1 Cells and Preparation of Plasma Membranes

P388.D1 cells are a mouse monocyte-macrophage like cell line. They were cultured in L-15 medium supplemented with 2 mM L-glutamine, 10% CS, 10 U penicillin, 100 g/ml streptomycin (all reagents from Sigma). Cells were split 1:4 every 3-4 days to maintain a cell density of 2-8×10⁵ per ml. A fresh aliquot of cells was thawed from liquid nitrogen every 6 weeks. Plasma membrane fractions were prepared as described in Example 2.

Competition ELISA

P388 plasma membranes (50 μl per well) were coated at 10 μg/ml in PBS onto Falcon 96-well plates overnight at 4° C. The method is otherwise essentially as described Example 16.

The results for 3 clones, I079E02, I082H08 and I099D03 are shown in FIG. 16. All 3 scFvs recognize P388 plasma membranes. This binding is competed out in the presence of HIS-tagged B Lymphocyte Stimulator (HIS-B Lymphocyte Stimulator) but not in the presence of biotinylated B Lymphocyte Stimulator (bio-B Lymphocyte Stimulator). This confirms that these scFvs also recognize the membrane-bound form but not the soluble form of mouse B Lymphocyte Stimulator.

Example 20 Conversion of scFvs to IgG1 Format

The VH domain and the VL domains of scFvs that we wished to convert into IgG molecules were cloned into vectors containing the nucleotide sequences of the appropriate heavy (human IgG1) or light chain (human kappa or human lambda) constant regions such that a complete heavy or light chain molecule could be expressed from these vectors when transfected into an appropriate host cell. Further, when cloned heavy and light chains are both expressed in one cell line (from either one or two vectors), they can assemble into a complete functional antibody molecule that is secreted into the cell culture medium. Methods for converting scFvs into conventional antibody molecules are well known within the art.

Generation of NS0 Cell Lines Expressing Anti-B Lymphocyte Stimulator Antibodies (IgG1)

Plasmids containing the heavy and light chains were separately linearized using the Pvu I restriction enzyme. The linearized DNAs were purified by phenol-chloroform extraction followed by ethanol precipitation and then resuspended in H₂O, NS0 cells (10⁷) from a growing culture were electroporated (0.25 kV and 975 μF) in PBS with 12.5 μg linearized heavy chain plasmid DNA and 37.5 μg linearized light chain DNA. The cells were washed in 20 ml non-selective medium (10% FCS in DMEM supplemented with 6 mM glutamine, amino acids and penicillin/streptomycin) and then transferred in 12.5 ml medium into a T75 cm² flask and incubated overnight at 37° C., 5% CO₂/air. The day after transfection the cells were resuspended in selective medium containing 1 mg/ml geneticin and dispensed into 5×96-well plates at 200 μl/well. After 18 days at 37° C. (5% CO₂/air) the colony supernatants were screened by an ELISA that detects assembled human IgG in order to identify colonies expressing IgG. Approximately twenty positive colonies were expanded and adapted to growth in serum-free, selective medium. Duplicate T25 cm² flasks were set up. Cells from one flask were frozen down as a stock and cells in the second flask were grown to saturation. The productivity of the saturated cultures was assessed by ELISA. The highest producing cell lines were then selected for large-scale antibody production.

The above procedure is exemplified for the I006D08 anti-B Lymphocyte Stimulator antibody constructs. Following electroporation and selection of NS0 cells, supernatants from ninety-three wells each containing a single colony were screened by ELISA to detect assembled IgG1, antibody. Twenty-seven of the supernatants were identified as containing IgG. The colonies from 24 of the positive wells were transferred to 1 ml selective medium in a 24-well plate and allowed to grow for 2 days. The 1 ml cultures of cells were then added to 4 ml selective medium containing reduced serum (0.5% FCS) in a T25 cm² flask. When the cultures reached confluency 1 ml cells were diluted in 4 ml selective, serum-free medium in a T25 cm² flask. At confluency this subculture regime was repeated again. Finally 1 ml cells from the culture containing 0.1% FCS was diluted with 9 ml serum-free, selective medium and divided into 2×T25 cm² to form the saturated and stock cultures. The stock cultures were frozen down and stored in liquid nitrogen once the cultures were confluent. The saturation culture was grown until the viability of the culture was <10%. Twenty-three out of the 24 colonies originally expanded were successfully adapted to growth in serum-free medium. The productivity of these serum-free adapted cell lines ranged from 0.3 to 17 μg/ml by ELISA quantification of the saturated, 5 ml serum-free cultures. The I006D08-32 cell line produced 17 μg/ml.

Large-Scale IgG Production

The highest-producing cell lines were revived from frozen stocks and then expanded to 400 ml in selective, serum-free medium in 2 liter roller bottles. The cells were grown at 37° C. and rolled at 4 rpm with the headspace being re-equilibrated with 5% CO₂/air every 2-3 days. Finally the culture was expanded to a 4 liter volume by the addition of serum-free medium without selection (400 ml per 2 liter roller bottle). The cultures were then grown to saturation.

This procedure is exemplified by the production of I006D08 antibody from the I006D08-32 cell line. The frozen stock of I006D08-32 was revived into a T25 cm² containing 5 ml serum-free medium containing 1 mg/ml geneticin and grown at 37° C. in 5% CO₂/air incubator. After two days growth the culture was diluted with 7.5 ml fresh medium and transferred to a T75 cm² flask. After a further three days in the incubator the cells were transferred to 130 ml selective medium and transferred to a 2 liter roller bottle. After three days growth the cells were diluted with 500 ml selective medium and split into 2×2 liter roller bottles. After another 2 days 100 ml fresh selective medium was added to each roller. Finally the next day the culture was expanded to a total volume of 4 liters with non-selective medium and divided into 10×2 liter roller bottles. After three days the medium was supplemented with 6 mM glutamine. The cells were grown for 17 days from the final subculture into a 4 liter volume. The cells grew up to 3×10⁶ cells/ml before viability declined to <0.2×10⁶ cells/ml. At this low viability the culture supernatants were harvested. ELISA analysis indicated that the culture supernatant contained 33 mg/ml IgG. Hence, the 4 liter culture contained 132 mg IgG.

IgG Purification

The purification of the IgG from the fermentation broth is performed using a combination of conventional techniques commonly used for antibody production. Typically the culture harvest is clarified to remove cells and cellular debris prior to starting the purification scheme. This would normally be achieved using either centrifugation or filtration of the harvest. Following clarification, the antibody would typically be captured and significantly purified using affinity chromatography on Protein A Sepharose. The antibody is bound to Protein A Sepharose at basic pH and, following washing of the matrix, is eluted by a reduction of the pH. Further purification of the antibody is then achieved by gel filtration. As well as removing components with different molecular weights from the antibody this step can also be used to buffer exchange into the desired final formulation buffer.

Purification of I006D08 IgG1

The harvest was clarified by sequential filtration through 0.5 μm and 0.22 μm filters. Clarified harvest was then applied to a column of recombinant Protein A Sepharose equilibrated at pH 8.0 and washed with the equilibration buffer. I006D08 antibody was eluted from the Protein A Sepharose by application of a buffer at pH3.5. The collected antibody containing eluate was then neutralized to pH 7.4 by the addition of pH 8.0 buffer. The neutralized eluate was concentrated by ultrafiltration using a 30 KDa cut off membrane. Concentrated material was then purified by Sephacryl S300HR gel filtration using phosphate buffered saline as the mobile phase. The final monomeric IgG1 fraction from the gel filtration column was then concentrated to the desired formulation concentration by ultrafiltration using a 30 KDa cut off membrane. The final product was filtered through a 0.22 μm filter.

Example 21 Antibody Neutralization of Murine Splenocyte Proliferation as Measured by 3HdT Incorporation

To determine if an antibody inhibited B Lymphocyte Stimulator mediated B cell proliferation, a splenocyte proliferation assay was performed Briefly, murine splenocytes were isolated by flushing spleen using a 25 g needle and 10 ml of complete medium (RPMI 1640 with 10% FBS containing 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM glutamine, 5×10⁻⁵M β-mercaptoethanol). The cells were passed through a 100 micron nylon filter to remove cell clumps. The cell suspension was then ficolled at 400×g for 25 minutes at room temperature (one 15 ml conical tube/spleen; 3 ml ficol, 10 ml cell suspension/spleen; Ficol 1083 from Sigma). The recovered cells were washed 3 times in complete medium and counted. Recovered cells were then diluted to a concentration of 3×10⁶/ml in complete medium containing a 3× concentration of SAC (3×=1:33,333 dilution of stock; stock is a 10% suspension of Staph. aureus (Cowan I strain) available from Calbiochem).

For each antibody, 50 microliters of antibody dilutions at 30 μg/ml, 3.0 μg/ml, and 0.3 μg/ml concentrations were aliquotted into individual wells of a 96 well plate in triplicate. Suitable positive controls, such as, for example monoclonal antibody 15C10, were also used. Antibody 15C10 is described in Moore et al., (1999), Science 285: 260-263 and WO0050597; 15C10 has also been deposited with the ATCC™ on Feb. 1, 2000 as ATCC™ Deposit No. PTA-1158. Medium containing no antibody (and human isotype controls (purchased commercially) when necessary) were used as negative controls.

B Lymphocyte Stimulator protein was diluted in complete medium to concentrations of 300 ng/ml, 90 ng/ml and 30 ng/ml. 50 microliters of each of the B Lymphocyte Stimulator dilutions were then added to the antibody dilution series in the plates. The plate containing the antibody and B Lymphocyte Stimulator dilutions are then incubated for 30 minutes at 37° C., 5% CO₂, after which 50 microliters of the splenocyte cell suspension containing SAC was added to all wells. The plates were then incubated for 72 hours (37° C., 5% CO₂).

After 72 hours, each well was supplemented with 50 μl of complete medium containing 0.5 μCi of 3H-thymidine (6.7 Ci/mM; Amersham) and cells were incubated for an additional 20-24 hours at (37° C., 5% CO₂). Following incubation cells were harvested using a Tomtec Cell Harvester and filters counted in a TopCount Scintillation counter (Packard).

Example 22 Human B Cell Proliferation Assay for In Vitro Screening of B Lymphocyte Stimulator Antagonist Molecules

The bioassay for assessing the effects of putative B Lymphocyte Stimulator antagonists was performed in triplicate in 96 well format by mixing equal volumes of B Lymphocyte Stimulator, responder cells, and putative antagonist each of which is prepared as a 3× stock reagent.

B-lymphocytes were purified from human tonsil by MACS (anti-CD3 depletion), washed, and resuspended in complete medium (CM) (RPMI 1640 with 10% FBS containing 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM glutamine, 5×10E-5 M beta-mercaptoethanol) at a concentration of 3×10e6 cells/mL. Staphylococcus aureus, Cowan I (SAC, CalBiochem) was added to cells at 3× concentration (3×=1:33,333 dilution of stock

Meanwhile, eight serial dilutions (3-fold) of potential antagonist were prepared in CM such that the diluted antagonists are at 3× the final concentrations to be tested in the assay. Antibodies are routinely tested starting at a final concentration of 10 ug/mL and going down to about 1.5 ng/mL.

Human rB Lymphocyte Stimulator was prepared in CM to 3× concentration (3×=300 ng/mL, 30 ng/mL, and 3 ng/mL) in CM. Potential inhibitors were routinely tested at several concentrations of B Lymphocyte Stimulator to avoid false negatives due to unexpectedly low affinity or antagonist concentration.

Fifty microliters of diluted antagonist and 50 uL of diluted B Lymphocyte Stimulator were added to the putative antagonist dilution series.

Cells were then incubated for 72 hours (37° C., 5% CO₂) in a fully humidified chamber. After 72 hrs., the cells were supplemented with 0.5 μCi/well 3H-thymidine (6.7 Ci/mmol) and incubated for an additional 24 hours. Plates were harvested using a Tomtec Cell Harvester and filters counted in a TopCount Scintillation counter (Packard).

Example 23 Characterization of I006D08 and I116A01

I116A01 (SEQ ID NO:327) is a CDR3 mutant of the I006D08 (SEQ ID NO:2). The I116A01 scFv was subsequently isolated from a single chain phage display library containing a repertoire of I006D08 mutations generated by randomization of the last 6 amino acids of the heavy chain CDR3 region of I006D08. The I116A01 scFv contains 7 amino acid differences from the parental I006D08 sequence, 1 change in the VH framework 3 region, 5 changes in the VH CDR3, and 1 change in the VLCDR3. Each of the assays described herein were performed using whole IgG1 molecules comprising the VH and VL regions of I006D08 and I116A01.

Binding Studies of I006D08 and I116A01

Equilibrium dissociation constants (Kd) for I116A01 and I006D08 binding to B Lymphocyte Stimulator were assessed using BIAcore technology (BIAcore, Uppsala, Sweden). Briefly, antibodies were captured on low-density Protein A surfaces and B Lymphocyte Stimulator was passed over at various flow rates. The binding of B Lymphocyte Stimulator to immobilized antibody was detected by changes in surface charge that were proportional to the amount of bound B Lymphocyte Stimulator. Binding values were calculated using software provided by the manufacturer.

I116A01 was determined to have an equilibrium dissociation constant, Kd, of 267±70 pM (mean±SD, n=3). The Kd value of I006D08, the parental antibody, was determined to be 498±151 pM (mean±SD, n=3). This was approximately 2-fold higher than the Kd obtained for I116A01. The difference in affinity can be attributed to a 2-fold higher off rate (kd, 1/s) of I006D08 compared with I116A01, the on rates (ka, 1/Ms) being essentially equal (Table 9).

TABLE 9 Kinetic parameters for I116A01 and I006D08 binding to B Lymphocyte Stimulator ka, 1/Ms kd, 1/s Kd, pM Mab Mean ± SD Mean ± SD Mean ± SD I116A01 1.26 ± 0.14 × 10⁶ 3.32 ± 0.63 × 10⁻⁴ 267 ± 70  I006D08 1.43 ± 0.13 × 10⁶  5.8 ± 0.24 × 10⁻⁴ 498 ± 151

The Kd values obtained for the B Lymphocyte Stimulator antibodies indicate that these antibodies bind to B Lymphocyte Stimulator with high affinity and possess a slow dissociation or off rate.

The binding characteristics of I116A01 and I006D08 were investigated further using a competitive binding ELISA in which B Lymphocyte Stimulator was immobilized directly on the surface of the well to compare epitopes. Biotinylated I006D08 binding was inhibited in a dose-dependent manner by the addition of unlabeled I006D08 or I116A01 suggesting that these antibodies recognize overlapping or identical epitopes on B Lymphocyte Stimulator.

Ability of I006D08 and I116A01 to Inhibit B Lymphocyte Stimulator-Induced Splenocyte Proliferation

The ability of I006D08 and I116A01 to inhibit B Lymphocyte Stimulator-induced splenocyte proliferation was tested using the assay described in Example 21 with slight modifications. In particular, the assay was modified such that the final concentration of B Lymphocyte Stimulator in the assay was 3 ng/ml and the final dilution factor of SAC was 1:100,000. Using this assay it was shown that both antibodies I006D08 and I116A01 were each found to inhibit murine splenocyte proliferation induced by both human (aa134-285 of SEQ ID NO:2) and murine soluble B Lymphocyte Stimulator. The IC₅₀ values for the inhibition of human B Lymphocyte Stimulator induced-proliferation of murine B cells was 0.09 nM±0.01 nM and 0.06 nM±0.01 nM, respectively.

Additional Characterization of I006D08 and I116A01

I006D08 and I116A01 were identified herein as antibodies that bind both the membrane bound and soluble forms of B Lymphocyte Stimulator. The ability to bind the membrane bound form of B Lymphocyte Stimulator was determined according to the assays described above, e.g., ability to bind HIS-tagged B Lymphocyte Stimulator and ability to bind U937 and/or P388 membrane preparations in an ELISA assay. Further characterization of antibodies I006D08 and I116A01 reveals that these antibodies bind recombinant B Lymphocyte Stimulator polypeptides expressed on the surface of a 293T cell. On the other hand, these antibodies are not able to bind naturally occurring B Lymphocyte Stimulator (non-recombinant) expressed on the surface of a cell, e.g., K-562 cells (ATCC™ #CCL-243) or U-937 cells (ATCC™ #CRL-1593.2). Thus, 1006D08 and I116A01 bind the soluble form of B Lymphocyte Stimulator. I006D08 and I116A01 also bind the membrane bound form of B Lymphocyte Stimulator (as determined by their ability to bind HIS-tagged B Lymphocyte Stimulator and their ability to bind U937 and/or P388 membrane preparations in an ELISA assay). I006D08 and I116A01 also bind recombinant B Lymphocyte Stimulator expressed on the surface of a 293T transfected fibroblast cell line, but do not bind naturally occurring B Lymphocyte Stimulator protein on the surface of a K-562 or U-937 cell.

Example 24 In Vivo Studies of Anti-B Lymphocyte Stimulator Antibodies

Overexpression of B Lymphocyte Stimulator in transgenic animals results in an autoimmune phenoptype including increased numbers of mature B cells (leading to enlarged spleens), increased levels of serum immunoglobulins, as well as increased levels of autoantibodies (e.g., anti-dsDNA antibodies) (McKay et al., (1999), The Journal of Experimental Medicine 190:1697-1710 and Gross et al., (2000) Nature 404:995-999, both of which are hereby incorporated by reference in their entireties.) Similar effects are observed when recombinant B Lymphocyte Stimulator is administered to animals such as mice and monkeys. The ability of anti-B Lymphocyte Stimulator antibodies of the invention to reduce or prevent these B Lymphocyte Stimulator induced effects may be tested through either co-administration or sequential admintration of soluble B Lymphocyte Stimulator and anti-B Lymphocyte Stimulator antibodies of the invention in an animal. An anti-B Lymphocyte Stimulator antibody of the invention would be considered neutralizing if it prevented or reduced B Lymphocyte Stimulator-induced increases in B cell numbers (resulting in lack of, or less, enlargement of spleen), and serum immunogloblins, and in particular serum autoantibodies.

Example 25 Antibody Production and Purification

The following example describes a large scale antibody production and purification methods that can be used to make antibodies of the present invention. One of skill in the art will be aware of routine modifications to the protocol described below, for example, as regards column choice, column, loading, wash, and elution buffers, and pH.

Cell Culture Scale-Up and Antibody Production

A serum-free and animal source-free growth medium is used from thawing cells through scale-up to the production bioreactor. The growth medium is prepared by adding 1 kilogram AGT powder granules preformulated with GS supplement to water for injection (WFI) quality water making a total of 51 kilograms CD hybridoma medium. To the CD hyrbridoma medium, 1 mL/kg cholesterol (synthetic) lipid concentrate (1000×). The AGT powder granules preformulated with GS supplement for preparing the CD hybridoma medium and the synthetic cholesterol lipid concentrate and are available from Invitrogen Corp., Carlsbad, Calif. The medium, hereinafter referred to as INV-CDH medium, is stored at 2-8° C. until use.

Thawing Cells from into Spinner Flask

Approximately 48×10⁶ NS0 cells engineered to express an antibody of the invention, preferably in whole IgG1 format, are thawed at 37° C. in a water bath. The cells are transferred into a 250 mL spinner flask to yield approximately a 150 mL working volume with an inoculation density of approximately 3.2×10⁵ cells/mL. The spinner flask(s) is then placed on magnetic stirrers in a humidified CO₂ incubator at 37° C. with 5% CO₂ for 4 days. The agitation rate for the spinner flask is 80 rpm.

First Expansion(s) of Culture in Spinner Flask

The culture is aseptically expanded into a 1000 mL spinner flask to give approximately 600 mL working volume, at an inoculation cell density of approximately 2.5×10⁵ cells/mL. The spinner flask is then placed on magnetic stirrers in a humidified CO₂ incubator at 37° C. with 5% CO₂ for 3 days. The agitation rate for the spinner flask is 80 rpm.

The culture is again expanded aseptically into two 3000 mL spinner flasks to give approximately 2×2000 mL working volume, at an inoculation cell density of approximately 3.0×10⁵ cells/mL. The spinner flask is then placed on magnetic stirrers in a humidified CO₂ incubator at 37° C. with 5% CO₂ for 3 days. The agitation rate for the spinner flasks is 80 rpm. At the same time, a reserve culture is created in a fresh 250 mL spinner flask. The reserve culture may be used as the seed for another lot as needed.

Seed Culture (25 Liters)

The 25 liter seed bioreactor is equipped with one impeller for mixing, a dissolved oxygen probe, a temperature probe, a pH probe, aseptic sampling and additional systems. The first step of the cell cultivation process is the addition of INV-CDH medium into the bioreactor. After the medium temperature reaches 37±0.5° C., the dissolved oxygen (DO) and pH levels are stabilized by addition of N₂ and CO₂ to decrease dissolved oxygen concentration to 25 to 75% air saturation, and obtain a pH of 7.20±0.2. The agitation rate is 70 rpm. The pooled cell culture is transferred aseptically to the 25 liter seed bioreactor containing 21 liters of sterile INV-CDH medium. During the cultivation process the temperature is maintained via a plant stem and chilled glycol, which regulate the temperature of the circulating jacket. The oxygen concentration is maintained via oxygen and nitrogen sparging and surface aeration, and pH is controlled by addition of CO₂ gas to lower the pH. The cultivation period is 4 days. The bioreactor air/gas inlets and vent lines are protected by hydrophobic 0.2 μm filters.

Seed Culture (200 Liters)

The 200 L seed bioreactor is equipped with 1 impeller for mixing, a dissolved oxygen probe, a temperature probe, a pH probe, aseptic sampling and additional systems. The first step of the cell cultivation process is the addition of INV-CDH medium into the bioreactor. After the medium temperature reaches 37±0.5° C., the DO and pH levels are stabilized by addition of N₂ and CO₂ to decrease dissolved oxygen concentration to 25 to 75% air saturation, and obtain a pH of 7.20±0.2. The agitation rate is 35 rpm. The 25 L seed culture is transferred aseptically to the 200 L seed bioreactor containing 175 L sterile medium. During the cultivation process the temperature is maintained via plant steam and chilled glycol which regulate the temperature of the circulating jacket. The oxygen concentration is maintained via oxygen and nitrogen sparging and surface aeration, and pH is controlled by addition of CO₂ gas to lower the pH. The cultivation period is 4 days. The bioreactor air/gas inlet and vent lines are protected by hydrophobic 0.2 μm filters.

Production Culture (1600 Liters)

The production bioreactor is equipped with 2 impellers for mixing, a dissolved oxygen probe, a temperature probe, a pH probe, aseptic sampling and additional systems. 600 L of INV-CDH medium is aseptically transferred into the 1600 L production bioreactor. After the medium temperature reaches 37±0.5° C., the DO and pH levels are stabilized by addition of N₂ and CO₂ to decrease dissolved oxygen concentration to 25 to 75% air saturation, and obtain a pH of 7.20±0.2. The agitation rate is 17 rpm. The 200 L seed culture is aseptically transferred into the production bioreactor. During the cultivation process the temperature is maintained via plant steam and chilled glycol which regulate the temperature of the circulating jacket, the oxygen concentration is maintained via oxygen and nitrohen sparging and surface aeration, and pH is controlled by addition of CO₂ gas to lower the pH. The bioreactor air/gas inlet and vent lines are protected by hydrophobic 0.2 μm filters. After 48 hours, 800 Liters of INV-CDH medium is transferred into the production bioreactor in order to obtain the final volume. The culture is cultivated in the production bioreactor 43 additional days.

Recovery and Purification Harvest of Cell Supernatant

Cell supernatant, (e.g., culture supernatant from NSO cells expressing antibodies of the invention) is harvested on day 5 or 6 post final feeding in the final production bioreactor. Cell culture broth temperature is cooled down to 15° C. in the recovery vessel at the time of harvest and maintained at that temperature during the recovery. Centrifugation followed by depth filtration is used for cell removal and antibody recovery. The filtration process train consists of 0.1 μm nominal rated depth filter and 0.2 μm membrane fileters connected in series. A constant flow rate of 13±1 L/min is maintained during the operation. The 0.2 μm filtered culture supernatant is collected in a process vessel, warned to 20° C. and transferred to purification. The purification process is conducted at 22 to 26° C.

Chromatography on rProtein A Sepharose FF Column

The culture supernatant is loaded onto rProtein-A Sepharose FF column (affinity chromatography, Amersham Pharmacia) or equivalent column that is equilibrated in 50 mM Tris-HCl, 0.5 M NaCl, pH 7.5. The rProtein-A Sepharose FF column is washed with 0.05 M sodium citrate, 0.15 M sodium chloride, pH 5.2 and eluted with 0.05 M sodium citrate, 0.15 M sodium chloride, pH 3.2. The elution is monitored by ultraviolet (UV) absorbance at 280 nm. The elution peak is collected, and analyzed by A₂₈₀ and SDS-PAGE.

Virus Inactivation

The eluate from the rProtein-A Sepharose FF column is adjusted with 1 M citric acid to pH 3.4±0.2 and allowed to stand for 45-60 minutes for viral inactivation. The solution is then readjusted to pH 5.4 with 1 M Tris base. The pH adjusted material is 0.2 μm filtered and stored at 20° C. up to 20 hours.

Chromatography on SP Sepharose FF Column

The inactivated material from the rProtein-A Sepharose FF column is diluted with water for injection (WFI) to a conductivity of 7 mS, and loaded onto a SP Sepharose FF column (cation exchanger, Amersham-Pharmacia), or equivalent column equilibrated with 50 mM sodium acetate, 30 mM sodium chloide, pH 5.4. The antibody is eluted from the SP column with 50 mM sodium acetate, 100 mM sodium chloride, pH 6.0. The elution is monitored by ultraviolet (UV) absorbance at 280 nm. The elution peak is collected and analyzed by A₂₈₀ and SDS-PAGE.

Virus Removal Filtration, Diafiltration and Concentration

The eluate from the SP Sepharose FF column is filtered through a sequentially connected 0.2 μm filter and a virus removal filter (Pall DV50 Ultipor® VF Filter System). The DV50 filtrate is stored at 20° C. up to 8 hours.

The DV50 filtrate is placed into a 30 kD molecular weight cut-off membrane device (Millipore Pellicon) to concentrate to a target concentration of 35-40 mg/mL. The concentrated material is diafiltered against 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, pH 6.5. The diafiltered material is monitored by A₂₈₀. The diafiltered bulk is 0.2 μm filtered and stored at 20° C. up to 8 hours.

Chromatography on Q Sepharose FF Column

The diafiltered antibody solution is passed over a Q Sepharose FF column (anion exchanger, Amersham-Pharmacia) or equivalent column equilibrated with 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, pH 6.5. The antibody is collected in the flow-through and monitored by A₂₈₀. The elution peak is collected and analyzed by A₂₈₀

Bulk Formulation, Filtration and Bulk Drug Substance Fill

Polysorbate 80 (1% stock solution in 10 mM sodium citrate, 1.9% glycine and 0.5% sucrose) is pre-filtered through a 0.2 μm filter and added to the antibody solution to a final concentration of 0.01%. The purified antibody is aseptically filtered under a laminar flow hood through a 0.2 μm filter and filled into polypropylene containers.

Storage of Bulk Drug Substance

The bulk drug substance is stored at 2-8° C. (short-term storage) or at or below −65° C. (long-term storage).

In Process Testing

In-process testing of bioreactor culture at harvest for each batch and in-process testing during the purification process are performed. The bioreactor is sampled aseptically and the culture is tested at various times throughout cultivation for cell density, viability and nutrient determination to ensure consistency of material being supplied for purification. The purification process is monitored at each step. Appearance is checked by vuisual inspection. The protein concentration is determined by Absorbance at 280 nm. The pH of the material is checked. Purity is checked, for example, by SDS-PAGE and size exclusion chromatography. An ELISA may be performed to check the ability of the antibody to bind its antigen. The biological activity of the antibody is also monitored. Residual DNA content, Endotoxin levels, and the bioburden (the number of viable organisms present in the antibody preparation) are all monitored and kept at or below standard acceptable levels. Additionally the oligosaccharide content may be analyzed; the peptide sequence of the antibody chains may also be analyzed using N-terminal sequencing and peptide mapping. Short and long-term studies of antibody stability may also be performed.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in this application is incorporated in their entireties herein by reference. Further, the sequences disclosed herein are also disclosed in the Sequence Listings of U.S. Provisional Application 60/212,210 filed Jun. 16, 2000 and U.S. Nonprovisional application Ser. No. 09/880,748 filed Jun. 15, 2001 the contents of which are herein incorporated in their entireties by reference.

The entire disclosure (including the specification, sequence listing, and drawings) of each of the following U.S. Provisional and Non-Provisional patent applications and International Patent Applications are herein incorporated by reference in their entireties: U.S. Provisional Application Ser. Nos. 60/543, 296 Filed Feb. 11, 2004; 60/580,347 fi9led Jun. 18, 2004; 60/331,469 filed Nov. 16, 2001; 60/340,817 filed Dec. 19, 2001; 60/212,210 filed Jun. 16, 2000; 60/240,816 filed Oct. 17, 2000; 60/276,248 filed Mar. 16, 2001; 60/277,379 filed Mar. 21, 2001; 60/293,499 filed May 25, 2001; and U.S. Nonprovisional application Ser. Nos. 10/293,418 and 09/880,748 filed Jun. 15, 2001; and International Patent Application Serial No. PCT/US01/19110 filed Jun. 15, 2001.

TABLE 1 scFvs that Immunospecifically Bind to B Lymphocyte Stimulator scFv SEQ  AAs of VL AAs of VL AAs of VL Clone ID ID NO AAs of VL CDR1 CDR2 CDR3 AAs of VH I003F12S 1 138-248 160-173 189-195 228-237 1-122 I006D08 2 141-249 163-173 189-195 228-238 1-123 I008A11 3 144-254 166-179 195-201 234-243 1-128 I017D10 4 148-255 169-179 195-201 234-244 1-132 I022D01 5 142-249 163-173 189-195 228-238 1-126 I031F02 6 138-251 160-173 189-195 228-240 1-121 I050A12 7 142-250 164-174 190-196 229-239 1-124 I051C04 8 146-256 168-181 197-203 236-245 1-129 I050B11 9 143-251 166-177 193-199 232-240 1-125 I050B11-01 10 143-251 166-177 193-199 232-240 1-125 I050B11-02 11 143-251 166-177 193-199 232-240 1-125 I050B11-03 12 143-251 166-177 193-199 232-240 1-125 I050B11-04 13 143-251 166-177 193-199 232-240 1-125 I050B11-05 14 143-251 166-177 193-199 232-240 1-125 I050B11-06 15 143-251 166-177 193-199 232-240 1-125 I050B11-07 16 143-251 166-177 193-199 232-240 1-125 I050B11-08 17 143-251 166-177 193-199 232-240 1-125 I050B11-09 18 143-251 166-177 193-199 232-240 1-125 I050B11-10 19 143-251 166-177 193-199 232-240 1-125 I050B11-11 20 143-251 166-177 193-199 232-240 1-125 I050B11-12 21 143-251 166-177 193-199 232-240 1-125 I050B11-13 22 143-251 166-177 193-199 232-240 1-125 I050B11-14 23 143-251 166-177 193-199 232-240 1-125 I050B11-15 24 143-251 166-177 193-199 232-240 1-125 I050B11-16 25 143-251 166-177 193-199 232-240 1-125 I050B11-17 26 143-251 166-177 193-199 232-240 1-125 I050B11-18 27 143-251 166-177 193-199 232-240 1-125 I050B11-19 28 143-251 166-177 193-199 232-240 1-125 I050B11-20 29 143-251 166-177 193-199 232-240 1-125 I050B11-21 30 143-251 166-177 193-199 232-240 1-125 I050B11-22 31 143-251 166-177 193-199 232-240 1-125 I050B11-23 32 143-251 166-177 193-199 232-240 1-125 I050B11-24 33 143-251 166-177 193-199 232-240 1-125 I050B11-25 34 143-251 166-177 193-199 232-240 1-125 I050B11-26 35 143-251 166-177 193-199 232-240 1-125 I050B11-27 36 143-251 166-177 193-199 232-240 1-125 I050B11-28 37 143-251 166-177 193-199 232-240 1-125 I093D03 38 143-251 166-177 193-199 232-240 1-125 I093D09 39 143-251 166-177 193-199 232-240 1-125 I093G08 40 143-251 166-177 193-199 232-240 1-125 I097D11 41 143-251 166-177 193-199 232-240 1-125 I101A04 42 143-251 166-177 193-199 232-240 1-125 I101B01 43 143-251 166-177 193-199 232-240 1-125 I102A02 44 143-251 166-177 193-199 232-240 1-125 I102E01 45 143-251 166-177 193-199 232-240 1-125 I102G06 46 143-251 166-177 193-199 232-240 1-125 I087A07 47 143-251 166-177 193-199 232-240 1-125 I087A08 48 142-250 165-176 192-198 231-239 1-124 I087A09 49 142-250 165-176 192-198 231-239 1-124 I087B02 50 142-250 165-176 192-198 231-239 1-124 I087B03 51 142-250 165-176 192-198 231-239 1-124 I087B04 52 142-250 165-176 192-198 231-239 1-124 I087B05 53 142-250 165-176 192-198 231-239 1-124 I087B06 54 142-250 165-176 192-198 231-239 1-124 I087B08 55 143-251 166-177 193-199 232-240 1-125 I087B09 56 142-250 165-176 192-198 231-239 1-124 I087C02 57 142-250 165-176 192-198 231-239 1-124 I087C05 58 142-250 165-176 192-198 231-239 1-124 I087C06 59 142-250 165-176 192-198 231-239 1-124 I087C07 60 142-250 165-176 192-198 231-239 1-124 I087C08 61 143-251 166-177 193-199 232-240 1-125 I087D01 62 142-250 165-176 192-198 231-239 1-124 I087D02 63 142-250 165-176 192-198 231-239 1-124 I087D03 64 143-251 166-177 193-199 232-240 1-125 I087D05 65 143-251 166-177 193-199 232-240 1-125 I087D07 66 142-250 165-176 192-198 231-239 1-124 I087D09 67 143-251 166-177 193-199 232-240 1-125 I087E04 68 143-251 166-177 193-199 232-240 1-125 I087E05 69 142-250 165-176 192-198 231-239 1-124 I087E10 70 143-251 166-177 193-199 232-240 1-125 I087F02 71 143-251 166-177 193-199 232-240 1-125 I087F04 72 143-251 166-177 193-199 232-240 1-125 I087F05 73 142-250 165-176 192-198 231-239 1-124 I087F07 74 142-250 165-176 192-198 231-239 1-124 I087F08 75 142-250 165-176 192-198 231-239 1-124 I087F09 76 142-250 165-176 192-198 231-239 1-124 I087G05 77 142-250 165-176 192-198 231-239 1-124 I087G06 78 142-250 165-176 192-198 231-239 1-124 I087G07 79 143-251 166-177 193-199 232-240 1-125 I087G09 80 142-250 165-176 192-198 231-239 1-124 I087G10 81 142-250 165-176 192-198 231-239 1-124 I087H02 82 137-244 160-170 186-192 225-233 1-121 I088A01 83 143-251 166-177 193-199 232-240 1-125 I088A03 84 143-251 166-177 193-199 232-240 1-125 I088A04 85 143-251 166-177 193-199 232-240 1-125 I088A08 86 143-251 166-177 193-199 232-240 1-125 I088A09 87 143-251 166-177 193-199 232-240 1-125 I088A10 88 143-251 166-177 193-199 232-240 1-125 I088A11 89 143-251 166-177 193-199 232-240 1-125 I088A12 90 143-251 166-177 193-199 232-240 1-125 I088B01 91 143-251 166-177 193-199 232-240 1-125 I088B02 92 143-251 166-177 193-199 232-240 1-125 I088B03 93 143-251 166-177 193-199 232-240 1-125 I088B05 94 143-251 166-177 193-199 232-240 1-125 I088B06 95 143-251 166-177 193-199 232-240 1-125 I088B07 96 143-251 166-177 193-199 232-240 1-125 I088B08 97 143-251 166-177 193-199 232-240 1-125 I088B09 98 143-251 166-177 193-199 232-240 1-125 I088B10 99 143-251 166-177 193-199 232-240 1-125 I088B12 100 142-250 165-176 192-198 231-239 1-124 I088C01 101 143-251 166-177 193-199 232-240 1-125 I088C03 102 143-251 166-177 193-199 232-240 1-125 I088C09 103 143-251 166-177 193-199 232-240 1-125 I088C12 104 143-251 166-177 193-199 232-240 1-125 I088D01 105 143-251 166-177 193-199 232-240 1-125 I088D03 106 143-251 166-177 193-199 232-240 1-125 I088D04 107 142-250 165-176 192-198 231-239 1-124 I088D07 108 143-251 166-177 193-199 232-240 1-125 I088D08 109 143-251 166-177 193-199 232-240 1-125 I088D11 110 143-251 166-177 193-199 232-240 1-125 I088E01 111 140-248 163-174 190-196 229-237 1-122 I088E02 112 143-251 166-177 193-199 232-240 1-125 I088E03 113 143-251 166-177 193-199 232-240 1-125 I088E04 114 143-251 166-177 193-199 232-240 1-125 I088E08 115 143-251 166-177 193-199 232-240 1-125 I088E10 116 143-251 166-177 193-199 232-240 1-125 I088E11 117 143-251 166-177 193-199 232-240 1-125 I088F07 118 143-251 166-177 193-199 232-240 1-125 I088G02 119 143-251 166-177 193-199 232-240 1-125 I088G03 120 143-251 166-177 193-199 232-240 1-125 I088G07 121 143-251 166-177 193-199 232-240 1-125 I088G09 122 143-251 166-177 193-199 232-240 1-125 I088G10 123 143-251 166-177 193-199 232-240 1-125 I088H05 124 143-251 166-177 193-199 232-240 1-125 I088H07 125 143-251 166-177 193-199 232-240 1-125 I092A03 126 143-251 166-177 193-199 232-240 1-125 I092A05 127 143-251 166-177 193-199 232-240 1-125 I092A06 128 143-251 166-177 193-199 232-240 1-125 I092A08 129 143-251 166-177 193-199 232-240 1-125 I092A10 130 143-251 166-177 193-199 232-240 1-125 I092A11 131 143-251 166-177 193-199 232-240 1-125 I092B01 132 143-251 166-177 193-199 232-240 1-125 I092B02 133 143-251 166-177 193-199 232-240 1-125 I092B04 134 143-251 166-177 193-199 232-240 1-125 I092B05 135 143-251 166-177 193-199 232-240 1-125 I092B10 136 143-251 166-177 193-199 232-240 1-125 I092B12 137 143-251 166-177 193-199 232-240 1-125 I092C01 138 143-251 166-177 193-199 232-240 1-125 I092C02 139 143-251 166-177 193-199 232-240 1-125 I092C07 140 142-250 165-176 192-198 231-239 1-124 I092C08 141 143-251 166-177 193-199 232-240 1-125 I092C12 142 143-251 166-177 193-199 232-240 1-125 I092D01 143 143-251 166-177 193-199 232-240 1-125 I092D07 144 143-251 166-177 193-199 232-240 1-125 I092D09 145 143-251 166-177 193-199 232-240 1-125 I092D10 146 143-251 166-177 193-199 232-240 1-125 I092D11 147 143-251 166-177 193-199 232-240 1-125 I092E01 148 143-251 166-177 193-199 232-240 1-125 I092E03 149 143-251 166-177 193-199 232-240 1-125 I092E04 150 143-251 166-177 193-199 232-240 1-125 I092E07 151 143-251 166-177 193-199 232-240 1-125 I092E10 152 143-251 166-177 193-199 232-240 1-125 I092E11 153 143-251 166-177 193-199 232-240 1-125 I092F01 154 143-251 166-177 193-199 232-240 1-125 I092F02 155 143-251 166-177 193-199 232-240 1-125 I092F05 156 143-251 166-177 193-199 232-240 1-125 I092F07 157 143-251 166-177 193-199 232-240 1-125 I092F08 158 143-251 166-177 193-199 232-240 1-125 I092F11 159 143-251 166-177 193-199 232-240 1-125 I092F12 160 143-251 166-177 193-199 232-240 1-125 I092G01 161 143-251 166-177 193-199 232-240 1-125 I092G05 162 143-251 166-177 193-199 232-240 1-125 I092G10 163 143-251 166-177 193-199 232-240 1-125 I092H01 164 137-244 160-170 186-192 225-233 1-121 I093A06 165 143-251 166-177 193-199 232-240 1-125 I093A09 166 143-251 166-177 193-199 232-240 1-125 I093A11 167 143-251 166-177 193-199 232-240 1-125 I093A12 168 143-251 166-177 193-199 232-240 1-125 I093B02 169 143-251 166-177 193-199 232-240 1-125 I093B05 170 143-251 166-177 193-199 232-240 1-125 I093B06 171 143-251 166-177 193-199 232-240 1-125 I093B09 172 143-251 166-177 193-199 232-240 1-125 I093B12 173 143-251 166-177 193-199 232-240 1-125 I093C02 174 143-251 166-177 193-199 232-240 1-125 I093C03 175 143-251 166-177 193-199 232-240 1-125 I093C05 176 143-251 166-177 193-199 232-240 1-125 I093D05 177 143-251 166-177 193-199 232-240 1-125 I093D08 178 143-251 166-177 193-199 232-240 1-125 I093D10 179 143-251 166-177 193-199 232-240 1-125 I093D12 180 143-251 166-177 193-199 232-240 1-125 I093E01 181 143-251 166-177 193-199 232-240 1-125 I093E02 182 143-251 166-177 193-199 232-240 1-125 I093E05 183 143-251 166-177 193-199 232-240 1-125 I093E08 184 143-251 166-177 193-199 232-240 1-125 I093E10 185 143-251 166-177 193-199 232-240 1-125 I093F01 186 143-251 166-177 193-199 232-240 1-125 I093F03 187 143-251 166-177 193-199 232-240 1-125 I093F05 188 143-251 166-177 193-199 232-240 1-125 I093F08 189 143-251 166-177 193-199 232-240 1-125 I093F11 190 143-251 166-177 193-199 232-240 1-125 I093G07 191 143-251 166-177 193-199 232-240 1-125 I093G11 192 143-251 166-177 193-199 232-240 1-125 I093G12 193 143-251 166-177 193-199 232-240 1-125 I093H06 194 143-251 166-177 193-199 232-240 1-125 I094A08 195 143-251 166-177 193-199 232-240 1-125 I094B07 196 143-251 166-177 193-199 232-240 1-125 I094B08 197 143-251 166-177 193-199 232-240 1-125 I094B12 198 143-251 166-177 193-199 232-240 1-125 I094C11 199 143-251 166-177 193-199 232-240 1-125 I094C12 200 143-251 166-177 193-199 232-240 1-125 I094D06 201 143-251 166-177 193-199 232-240 1-125 I094D07 202 143-251 166-177 193-199 232-240 1-125 I094D08 203 143-251 166-177 193-199 232-240 1-125 I094D09 204 143-251 166-177 193-199 232-240 1-125 I094D10 205 143-251 166-177 193-199 232-240 1-125 I094D11 206 143-251 166-177 193-199 232-240 1-125 I094E04 207 143-251 166-177 193-199 232-240 1-125 I094E08 208 143-251 166-177 193-199 232-240 1-125 I094F04 209 143-251 166-177 193-199 232-240 1-125 I094F05 210 143-251 166-177 193-199 232-240 1-125 I094F10 211 143-251 166-177 193-199 232-240 1-125 I094F11 212 143-251 166-177 193-199 232-240 1-125 I094F12 213 143-251 166-177 193-199 232-240 1-125 I094G06 214 143-251 166-177 193-199 232-240 1-125 I094G10 215 143-251 166-177 193-199 232-240 1-125 I095A04 216 143-251 166-177 193-199 232-240 1-125 I095A12 217 143-251 166-177 193-199 232-240 1-125 I095B04 218 143-251 166-177 193-199 232-240 1-125 I095B09 219 143-251 166-177 193-199 232-240 1-125 I095B10 220 143-251 166-177 193-199 232-240 1-125 I095C02 221 143-251 166-177 193-199 232-240 1-125 I095C05 222 143-251 166-177 193-199 232-240 1-125 I095C07 223 143-251 166-177 193-199 232-240 1-125 I095C08 224 143-251 166-177 193-199 232-240 1-125 I095C09 225 143-251 166-177 193-199 232-240 1-125 I095D01 226 143-251 166-177 193-199 232-240 1-125 I095D02 227 143-251 166-177 193-199 232-240 1-125 I095D03 228 143-251 166-177 193-199 232-240 1-125 I095D05 229 143-251 166-177 193-199 232-240 1-125 I095D09 230 143-251 166-177 193-199 232-240 1-125 I095E01 231 143-251 166-177 193-199 232-240 1-125 I095E05 232 143-251 166-177 193-199 232-240 1-125 I095E12 233 143-251 166-177 193-199 232-240 1-125 I095F06 234 143-251 166-177 193-199 232-240 1-125 I095F09 235 143-251 166-177 193-199 232-240 1-125 I095G06 236 143-251 166-177 193-199 232-240 1-125 I095G09 237 143-251 166-177 193-199 232-240 1-125 I095G11 238 143-251 166-177 193-199 232-240 1-125 I096A01 239 143-251 166-177 193-199 232-240 1-125 I096A10 240 143-251 166-177 193-199 232-240 1-125 I096B01 241 143-251 166-177 193-199 232-240 1-125 I096B03 242 143-251 166-177 193-199 232-240 1-125 I096C01 243 143-251 166-177 193-199 232-240 1-125 I096C06 244 143-251 166-177 193-199 232-240 1-125 I096C09 245 143-251 166-177 193-199 232-240 1-125 I096D01 246 143-251 166-177 193-199 232-240 1-125 I096D02 247 143-251 166-177 193-199 232-240 1-125 I096D05 248 143-251 166-177 193-199 232-240 1-125 I096D06 249 143-251 166-177 193-199 232-240 1-125 I096D09 250 143-251 166-177 193-199 232-240 1-125 I096E02 251 143-251 166-177 193-199 232-240 1-125 I096E06 252 143-251 166-177 193-199 232-240 1-125 I096E11 253 143-251 166-177 193-199 232-240 1-125 I096F02 254 143-251 166-177 193-199 232-240 1-125 I096G01 255 143-251 166-177 193-199 232-240 1-125 I096G02 256 143-251 166-177 193-199 232-240 1-125 I096G05 257 143-251 166-177 193-199 232-240 1-125 I096G07 258 143-251 166-177 193-199 232-240 1-125 I096G09 259 143-251 166-177 193-199 232-240 1-125 I096G12 260 143-251 166-177 193-199 232-240 1-125 I096H01 261 137-244 160-170 186-192 225-233 1-121 I097A04 262 143-251 166-177 193-199 232-240 1-125 I097A06 263 143-251 166-177 193-199 232-240 1-125 I097A09 264 143-251 166-177 193-199 232-240 1-125 I097B02 265 143-251 166-177 193-199 232-240 1-125 I097B09 266 143-251 166-177 193-199 232-240 1-125 I097B10 267 143-251 166-177 193-199 232-240 1-125 I097B11 268 143-251 166-177 193-199 232-240 1-125 I097C05 269 143-251 166-177 193-199 232-240 1-125 I097C09 270 143-251 166-177 193-199 232-240 1-125 I097C11 271 143-251 166-177 193-199 232-240 1-125 I097D05 272 143-251 166-177 193-199 232-240 1-125 I097D06 273 143-251 166-177 193-199 232-240 1-125 I097E01 274 143-251 166-177 193-199 232-240 1-125 I097E04 275 143-251 166-177 193-199 232-240 1-125 I097E08 276 143-251 166-177 193-199 232-240 1-125 I097E09 277 143-251 166-177 193-199 232-240 1-125 I097F09 278 143-251 166-177 193-199 232-240 1-125 I097G10 279 143-251 166-177 193-199 232-240 1-125 I097H02 280 137-244 160-170 186-192 225-233 1-121 I098A04 281 143-251 166-177 193-199 232-240 1-125 I098A05 282 143-251 166-177 193-199 232-240 1-125 I098B08 283 143-251 166-177 193-199 232-240 1-125 I098C01 284 143-251 166-177 193-199 232-240 1-125 I098C04 285 143-251 166-177 193-199 232-240 1-125 I098F11 286 143-251 166-177 193-199 232-240 1-125 I098F12 287 143-251 166-177 193-199 232-240 1-125 I098G02 288 143-251 166-177 193-199 232-240 1-125 I098G12 289 143-251 166-177 193-199 232-240 1-125 I098H05 290 143-251 166-177 193-199 232-240 1-125 I101A01 291 143-251 166-177 193-199 232-240 1-125 I101B04 292 143-251 166-177 193-199 232-240 1-125 I101B06 293 143-251 166-177 193-199 232-240 1-125 I101D04 294 143-251 166-177 193-199 232-240 1-125 I101D07 295 143-251 166-177 193-199 232-240 1-125 I101E09 296 143-251 166-177 193-199 232-240 1-125 I101E12 297 143-251 166-177 193-199 232-240 1-125 I101G02 298 143-251 166-177 193-199 232-240 1-125 I101G11 299 143-251 166-177 193-199 232-240 1-125 I102C03 300 143-251 166-177 193-199 232-240 1-125 I102E09 301 143-251 166-177 193-199 232-240 1-125 I102F02 302 143-251 166-177 193-199 232-240 1-125 I102G08 303 143-251 166-177 193-199 232-240 1-125 I102G09 304 143-251 166-177 193-199 232-240 1-125 I106A09 305 143-251 166-177 193-199 232-240 1-125 I106B02 306 143-251 166-177 193-199 232-240 1-125 I106B06 307 143-251 166-177 193-199 232-240 1-125 I106C07 308 143-251 166-177 193-199 232-240 1-125 I106E05 309 143-251 166-177 193-199 232-240 1-125 I106E12 310 143-251 166-177 193-199 232-240 1-125 I106G01 311 143-251 166-177 193-199 232-240 1-125 I106G03 312 143-251 166-177 193-199 232-240 1-125 I109B06 313 143-251 166-177 193-199 232-240 1-125 I109D12 314 143-251 166-177 193-199 232-240 1-125 I109E12 315 143-251 166-177 193-199 232-240 1-125 I109G06 316 143-251 166-177 193-199 232-240 1-125 I109H04 317 143-251 166-177 193-199 232-240 1-125 I110B03 318 143-251 166-177 193-199 232-240 1-125 I112D09 319 143-251 166-177 193-199 232-240 1-125 I112F10 320 143-251 166-177 193-199 232-240 1-125 I089F12 321 141-249 163-173 189-195 228-238 1-123 I105E12 322 141-249 163-173 189-195 228-238 1-123 I108D08 323 141-249 163-173 189-195 228-238 1-123 I108E06 324 141-249 163-173 189-195 228-238 1-123 I113E07 325 141-249 163-173 189-195 228-238 1-123 I114G05 326 141-249 163-173 189-195 228-238 1-123 I116A01 327 141-249 163-173 189-195 228-238 1-123 I116A09 328 141-249 163-173 189-195 228-238 1-123 I116C11 329 141-249 163-173 189-195 228-238 1-123 I085A01 330 141-249 163-173 189-195 228-238 1-123 I085A02 331 141-249 163-173 189-195 228-238 1-123 I085A03 332 141-249 163-173 189-195 228-238 1-123 I085A04 333 141-249 163-173 189-195 228-238 1-123 I085A05 334 141-249 163-173 189-195 228-238 1-123 I085A06 335 141-249 163-173 189-195 228-238 1-123 I085A07 336 141-249 163-173 189-195 228-238 1-123 I085A09 337 140-248 162-172 188-194 227-237 1-122 I085A10 338 141-249 163-173 189-195 228-238 1-123 I085A11 339 140-248 162-172 188-194 227-237 1-122 I085B01 340 141-249 163-173 189-195 228-238 1-123 I085B02 341 141-249 163-173 189-195 228-238 1-123 I085B03 342 141-249 163-173 189-195 228-238 1-123 I085B04 343 141-249 163-173 189-195 228-238 1-123 I085B05 344 141-249 163-173 189-195 228-238 1-123 I085B06 345 141-249 163-173 189-195 228-238 1-123 I085B07 346 141-249 163-173 189-195 228-238 1-123 I085B10 347 140-248 162-172 188-194 227-237 1-122 I085B12 348 141-249 163-173 189-195 228-238 1-123 I085C02 349 141-249 163-173 189-195 228-238 1-123 I085C03 350 141-249 163-173 189-195 228-238 1-123 I085C05 351 141-249 163-173 189-195 228-238 1-123 I085C06 352 141-249 163-173 189-195 228-238 1-123 I085C07 353 141-249 163-173 189-195 228-238 1-123 I085C09 354 140-248 162-172 188-194 227-237 1-122 I085C10 355 141-249 163-173 189-195 228-238 1-123 I085C12 356 141-249 163-173 189-195 228-238 1-123 I085D01 357 141-249 163-173 189-195 228-238 1-123 I085D02 358 141-249 163-173 189-195 228-238 1-123 I085D03 359 141-249 163-173 189-195 228-238 1-123 I085D04 360 141-249 163-173 189-195 228-238 1-123 I085D06 361 141-249 163-173 189-195 228-238 1-123 I085D07 362 141-249 163-173 189-195 228-238 1-123 I085D08 363 141-249 163-173 189-195 228-238 1-123 I085D09 364 141-249 163-173 189-195 228-238 1-123 I085D10 365 141-249 163-173 189-195 228-238 1-123 I085D11 366 141-249 163-173 189-195 228-238 1-123 I085D12 367 141-249 163-173 189-195 228-238 1-123 I085E01 368 141-249 163-173 189-195 228-238 1-123 I085E02 369 141-249 163-173 189-195 228-238 1-123 I085E07 370 141-249 163-173 189-195 228-238 1-123 I085E08 371 141-249 163-173 189-195 228-238 1-123 I085E09 372 141-249 163-173 189-195 228-238 1-123 I085E10 373 141-249 163-173 189-195 228-238 1-123 I085E11 374 141-249 163-173 189-195 228-238 1-123 I085E12 375 141-249 163-173 189-195 228-238 1-123 I085F01 376 141-249 163-173 189-195 228-238 1-123 I085F02 377 141-249 163-173 189-195 228-238 1-123 I085F03 378 141-249 163-173 189-195 228-238 1-123 I085F04 379 141-249 163-173 189-195 228-238 1-123 I085F05 380 141-249 163-173 189-195 228-238 1-123 I085F06 381 141-249 163-173 189-195 228-238 1-123 I085F07 382 141-249 163-173 189-195 228-238 1-123 I085F09 383 141-249 163-173 189-195 228-238 1-123 I085F10 384 141-249 163-173 189-195 228-238 1-123 I085F11 385 141-249 163-173 189-195 228-238 1-123 I085F12 386 141-249 163-173 189-195 228-238 1-123 I085G01 387 141-249 163-173 189-195 228-238 1-123 I085G02 388 141-249 163-173 189-195 228-238 1-123 I085G03 389 141-249 163-173 189-195 228-238 1-123 I085G04 390 141-249 163-173 189-195 228-238 1-123 I085G07 391 141-249 163-173 189-195 228-238 1-123 I085G08 392 141-249 163-173 189-195 228-238 1-123 I085G09 393 141-249 163-173 189-195 228-238 1-123 I085G10 394 140-248 162-172 188-194 227-237 1-122 I085G11 395 141-249 163-173 189-195 228-238 1-123 I085G12 396 141-249 163-173 189-195 228-238 1-123 I085H10 397 142-249 163-173 189-195 228-238 1-126 I086A03 398 141-249 163-173 189-195 228-238 1-123 I086A04 399 141-249 163-173 189-195 228-238 1-123 I086A05 400 141-249 163-173 189-195 228-238 1-123 I086A07 401 141-249 163-173 189-195 228-238 1-123 I086A09 402 141-249 163-173 189-195 228-238 1-123 I086A10 403 141-249 163-173 189-195 228-238 1-123 I086A11 404 141-249 163-173 189-195 228-238 1-123 I086A12 405 141-249 163-173 189-195 228-238 1-123 I086B02 406 141-249 163-173 189-195 228-238 1-123 I086B03 407 141-249 163-173 189-195 228-238 1-123 I086B05 408 141-249 163-173 189-195 228-238 1-123 I086B06 409 141-249 163-173 189-195 228-238 1-123 I086B07 410 141-249 163-173 189-195 228-238 1-123 I086B09 411 141-249 163-173 189-195 228-238 1-123 I086B10 412 141-249 163-173 189-195 228-238 1-123 I086B11 413 141-249 163-173 189-195 228-238 1-123 I086C03 414 141-249 163-173 189-195 228-238 1-123 I086C05 415 141-249 163-173 189-195 228-238 1-123 I086C07 416 141-249 163-173 189-195 228-238 1-123 I086C08 417 141-249 163-173 189-195 228-238 1-123 I086C09 418 141-249 163-173 189-195 228-238 1-123 I086C10 419 141-249 163-173 189-195 228-238 1-123 I086C11 420 141-249 163-173 189-195 228-238 1-123 I086C12 421 141-249 163-173 189-195 228-238 1-123 I086D01 422 141-249 163-173 189-195 228-238 1-123 I086D04 423 141-249 163-173 189-195 228-238 1-123 I086D05 424 141-249 163-173 189-195 228-238 1-123 I086D06 425 141-249 163-173 189-195 228-238 1-123 I086D07 426 141-249 163-173 189-195 228-238 1-123 I086D08 427 141-249 163-173 189-195 228-238 1-123 I086D09 428 141-249 163-173 189-195 228-238 1-123 I086D10 429 141-249 163-173 189-195 228-238 1-123 I086D11 430 141-249 163-173 189-195 228-238 1-123 I086D12 431 141-249 163-173 189-195 228-238 1-123 I086E02 432 141-249 163-173 189-195 228-238 1-123 I086E03 433 141-249 163-173 189-195 228-238 1-123 I086E05 434 141-249 163-173 189-195 228-238 1-123 I086E07 435 141-249 163-173 189-195 228-238 1-123 I086E08 436 141-249 163-173 189-195 228-238 1-123 I086E09 437 141-249 163-173 189-195 228-238 1-123 I086E10 438 141-249 163-173 189-195 228-238 1-123 I086E12 439 141-249 163-173 189-195 228-238 1-123 I086F02 440 141-249 163-173 189-195 228-238 1-123 I086F05 441 141-249 163-173 189-195 228-238 1-123 I086F08 442 141-249 163-173 189-195 228-238 1-123 I086F09 443 141-249 163-173 189-195 228-238 1-123 I086F11 444 141-249 163-173 189-195 228-238 1-123 I086G03 445 141-249 163-173 189-195 228-238 1-123 I086G04 446 141-249 163-173 189-195 228-238 1-123 I086G05 447 141-249 163-173 189-195 228-238 1-123 I086G06 448 141-249 163-173 189-195 228-238 1-123 I086G07 449 141-249 163-173 189-195 228-238 1-123 I086G09 450 141-249 163-173 189-195 228-238 1-123 I086G10 451 141-249 163-173 189-195 228-238 1-123 I086H05 452 141-249 163-173 189-195 228-238 1-123 I089A01 453 140-248 162-172 188-194 227-237 1-122 I089A03 454 141-249 163-173 189-195 228-238 1-123 I089A06 455 141-249 163-173 189-195 228-238 1-123 I089A07 456 141-249 163-173 189-195 228-238 1-123 I089A08 457 141-249 163-173 189-195 228-238 1-123 I089A10 458 141-249 163-173 189-195 228-238 1-123 I089A11 459 141-249 163-173 189-195 228-238 1-123 I089B01 460 141-249 163-173 189-195 228-238 1-123 I089B02 461 141-249 163-173 189-195 228-238 1-123 I089B03 462 141-249 163-173 189-195 228-238 1-123 I089B04 463 141-249 163-173 189-195 228-238 1-123 I089B05 464 141-249 163-173 189-195 228-238 1-123 I089B06 465 141-249 163-173 189-195 228-238 1-123 I089B07 466 141-249 163-173 189-195 228-238 1-123 I089B08 467 141-249 163-173 189-195 228-238 1-123 I089B09 468 141-249 163-173 189-195 228-238 1-123 I089B10 469 141-249 163-173 189-195 228-238 1-123 I089B11 470 141-249 163-173 189-195 228-238 1-123 I089C01 471 141-249 163-173 189-195 228-238 1-123 I089C02 472 141-249 163-173 189-195 228-238 1-123 I089C03 473 141-249 163-173 189-195 228-238 1-123 I089C05 474 141-249 163-173 189-195 228-238 1-123 I089C06 475 141-249 163-173 189-195 228-238 1-123 I089C07 476 141-249 163-173 189-195 228-238 1-123 I089C09 477 141-249 163-173 189-195 228-238 1-123 I089D01 478 141-249 163-173 189-195 228-238 1-123 I089D02 479 141-249 163-173 189-195 228-238 1-123 I089D03 480 141-249 163-173 189-195 228-238 1-123 I089D04 481 141-249 163-173 189-195 228-238 1-123 I089D05 482 141-249 163-173 189-195 228-238 1-123 I089D07 483 141-249 163-173 189-195 228-238 1-123 I089D08 484 140-248 162-172 188-194 227-237 1-122 I089D09 485 141-249 163-173 189-195 228-238 1-123 I089D11 486 141-249 163-173 189-195 228-238 1-123 I089E01 487 141-249 163-173 189-195 228-238 1-123 I089E02 488 141-249 163-173 189-195 228-238 1-123 I089E03 489 141-249 163-173 189-195 228-238 1-123 I089E04 490 141-249 163-173 189-195 228-238 1-123 I089E06 491 141-249 163-173 189-195 228-238 1-123 I089E09 492 141-249 163-173 189-195 228-238 1-123 I089E10 493 141-249 163-173 189-195 228-238 1-123 I089E11 494 141-249 163-173 189-195 228-238 1-123 I089F01 495 141-249 163-173 189-195 228-238 1-123 I089F03 496 141-249 163-173 189-195 228-238 1-123 I089F04 497 141-249 163-173 189-195 228-238 1-123 I089F05 498 141-249 163-173 189-195 228-238 1-123 I089F06 499 141-249 163-173 189-195 228-238 1-123 I089F08 500 141-249 163-173 189-195 228-238 1-123 I089F09 501 141-249 163-173 189-195 228-238 1-123 I089F10 502 141-249 163-173 189-195 228-238 1-123 I089F11 503 141-249 163-173 189-195 228-238 1-123 I089G01 504 141-249 163-173 189-195 228-238 1-123 I089G02 505 141-249 163-173 189-195 228-238 1-123 I089G03 506 141-249 163-173 189-195 228-238 1-123 I089G05 507 141-249 163-173 189-195 228-238 1-123 I089G06 508 141-249 163-173 189-195 228-238 1-123 I089G07 509 141-249 163-173 189-195 228-238 1-123 I089G08 510 141-249 163-173 189-195 228-238 1-123 I089G11 511 141-249 163-173 189-195 228-238 1-123 I089H10 512 142-249 163-173 189-195 228-238 1-126 I090A02 513 141-249 163-173 189-195 228-238 1-123 I090A03 514 141-249 163-173 189-195 228-238 1-123 I090A04 515 141-249 163-173 189-195 228-238 1-123 I090A05 516 141-249 163-173 189-195 228-238 1-123 I090A06 517 141-249 163-173 189-195 228-238 1-123 I090A07 518 141-249 163-173 189-195 228-238 1-123 I090A08 519 141-249 163-173 189-195 228-238 1-123 I090B01 520 141-249 163-173 189-195 228-238 1-123 I090B03 521 141-249 163-173 189-195 228-238 1-123 I090B04 522 141-249 163-173 189-195 228-238 1-123 I090B05 523 141-249 163-173 189-195 228-238 1-123 I090B06 524 141-249 163-173 189-195 228-238 1-123 I090B08 525 141-249 163-173 189-195 228-238 1-123 I090B11 526 141-249 163-173 189-195 228-238 1-123 I090B12 527 141-249 163-173 189-195 228-238 1-123 I090001 528 141-249 163-173 189-195 228-238 1-123 I0900O2 529 141-249 163-173 189-195 228-238 1-123 I0900O3 530 141-249 163-173 189-195 228-238 1-123 I090005 531 141-249 163-173 189-195 228-238 1-123 I090006 532 141-249 163-173 189-195 228-238 1-123 I090007 533 141-249 163-173 189-195 228-238 1-123 I090008 534 141-249 163-173 189-195 228-238 1-123 I090C10 535 141-249 163-173 189-195 228-238 1-123 I090D02 536 141-249 163-173 189-195 228-238 1-123 I090D03 537 141-249 163-173 189-195 228-238 1-123 I090D04 538 141-249 163-173 189-195 228-238 1-123 I090D05 539 141-249 163-173 189-195 228-238 1-123 I090D06 540 141-249 163-173 189-195 228-238 1-123 I090D07 541 141-249 163-173 189-195 228-238 1-123 I090D08 542 141-249 163-173 189-195 228-238 1-123 I090D09 543 141-249 163-173 189-195 228-238 1-123 I090D12 544 141-249 163-173 189-195 228-238 1-123 I090E04 545 141-249 163-173 189-195 228-238 1-123 I090E05 546 141-249 163-173 189-195 228-238 1-123 I090E06 547 141-249 163-173 189-195 228-238 1-123 I090E07 548 141-249 163-173 189-195 228-238 1-123 I090E09 549 141-249 163-173 189-195 228-238 1-123 I090E11 550 141-249 163-173 189-195 228-238 1-123 I090E12 551 141-249 163-173 189-195 228-238 1-123 I090F01 552 141-249 163-173 189-195 228-238 1-123 I090F02 553 141-249 163-173 189-195 228-238 1-123 I090F03 554 141-249 163-173 189-195 228-238 1-123 I090F04 555 141-249 163-173 189-195 228-238 1-123 I090F05 556 141-249 163-173 189-195 228-238 1-123 I090F06 557 141-249 163-173 189-195 228-238 1-123 I090F07 558 141-249 163-173 189-195 228-238 1-123 I090F08 559 141-249 163-173 189-195 228-238 1-123 I090F09 560 141-249 163-173 189-195 228-238 1-123 I090F10 561 141-249 163-173 189-195 228-238 1-123 I090F11 562 141-249 163-173 189-195 228-238 1-123 I090G01 563 141-249 163-173 189-195 228-238 1-123 I090G02 564 141-249 163-173 189-195 228-238 1-123 I090G04 565 141-249 163-173 189-195 228-238 1-123 I090G05 566 141-249 163-173 189-195 228-238 1-123 I090G06 567 141-249 163-173 189-195 228-238 1-123 I090G07 568 141-249 163-173 189-195 228-238 1-123 I090G08 569 141-249 163-173 189-195 228-238 1-123 I090G09 570 141-249 163-173 189-195 228-238 1-123 I090G10 571 141-249 163-173 189-195 228-238 1-123 I090G12 572 141-249 163-173 189-195 228-238 1-123 I091A02 573 141-249 163-173 189-195 228-238 1-123 I091A03 574 141-249 163-173 189-195 228-238 1-123 I091A06 575 141-249 163-173 189-195 228-238 1-123 I091A11 576 141-249 163-173 189-195 228-238 1-123 I091B01 577 141-249 163-173 189-195 228-238 1-123 I091B02 578 141-249 163-173 189-195 228-238 1-123 I091B04 579 141-249 163-173 189-195 228-238 1-123 I091B05 580 141-249 163-173 189-195 228-238 1-123 I091B07 581 141-249 163-173 189-195 228-238 1-123 I091B10 582 141-249 163-173 189-195 228-238 1-123 I091B11 583 141-249 163-173 189-195 228-238 1-123 I091B12 584 141-249 163-173 189-195 228-238 1-123 I091C02 585 141-249 163-173 189-195 228-238 1-123 I091C03 586 141-249 163-173 189-195 228-238 1-123 I091C04 587 141-249 163-173 189-195 228-238 1-123 I091C05 588 141-249 163-173 189-195 228-238 1-123 I091C06 589 141-249 163-173 189-195 228-238 1-123 I091C09 590 141-249 163-173 189-195 228-238 1-123 I091C11 591 141-249 163-173 189-195 228-238 1-123 I091C12 592 141-249 163-173 189-195 228-238 1-123 I091D01 593 141-249 163-173 189-195 228-238 1-123 I091D02 594 141-249 163-173 189-195 228-238 1-123 I091D04 595 141-249 163-173 189-195 228-238 1-123 I091D05 596 141-249 163-173 189-195 228-238 1-123 I091D06 597 140-248 162-172 188-194 227-237 1-122 I091D07 598 141-249 163-173 189-195 228-238 1-123 I091D09 599 141-249 163-173 189-195 228-238 1-123 I091E01 600 141-249 163-173 189-195 228-238 1-123 I091E02 601 141-249 163-173 189-195 228-238 1-123 I091E03 602 141-249 163-173 189-195 228-238 1-123 I091E04 603 141-249 163-173 189-195 228-238 1-123 I091E06 604 141-249 163-173 189-195 228-238 1-123 I091E07 605 141-249 163-173 189-195 228-238 1-123 I091E08 606 141-249 163-173 189-195 228-238 1-123 I091E09 607 141-249 163-173 189-195 228-238 1-123 I091E10 608 141-249 163-173 189-195 228-238 1-123 I091F01 609 141-249 163-173 189-195 228-238 1-123 I091F03 610 141-249 163-173 189-195 228-238 1-123 I091F05 611 141-249 163-173 189-195 228-238 1-123 I091F06 612 141-249 163-173 189-195 228-238 1-123 I091F07 613 141-249 163-173 189-195 228-238 1-123 I091F08 614 141-249 163-173 189-195 228-238 1-123 I091F09 615 141-249 163-173 189-195 228-238 1-123 I091F10 616 141-249 163-173 189-195 228-238 1-123 I091F11 617 141-249 163-173 189-195 228-238 1-123 I091F12 618 141-249 163-173 189-195 228-238 1-123 I091G01 619 141-249 163-173 189-195 228-238 1-123 I091G03 620 141-249 163-173 189-195 228-238 1-123 I091G04 621 141-249 163-173 189-195 228-238 1-123 I091G05 622 141-249 163-173 189-195 228-238 1-123 I091G06 623 140-248 162-172 188-194 227-237 1-122 I091G07 624 141-249 163-173 189-195 228-238 1-123 I091G09 625 141-249 163-173 189-195 228-238 1-123 I091G10 626 141-249 163-173 189-195 228-238 1-123 I091G11 627 141-249 163-173 189-195 228-238 1-123 I091G12 628 141-249 163-173 189-195 228-238 1-123 I104A01 629 141-249 163-173 189-195 228-238 1-123 I104A07 630 141-249 163-173 189-195 228-238 1-123 I104A08 631 141-249 163-173 189-195 228-238 1-123 I104A09 632 141-249 163-173 189-195 228-238 1-123 I104A10 633 141-249 163-173 189-195 228-238 1-123 I104A11 634 141-249 163-173 189-195 228-238 1-123 I104A12 635 141-249 163-173 189-195 228-238 1-123 I104B02 636 141-249 163-173 189-195 228-238 1-123 I104B04 637 141-249 163-173 189-195 228-238 1-123 I104B09 638 141-249 163-173 189-195 228-238 1-123 I104B11 639 141-249 163-173 189-195 228-238 1-123 I104C01 640 141-249 163-173 189-195 228-238 1-123 I104C04 641 141-249 163-173 189-195 228-238 1-123 I104C05 642 141-249 163-173 189-195 228-238 1-123 I104C06 643 140-248 162-172 188-194 227-237 1-122 I104C07 644 141-249 163-173 189-195 228-238 1-123 I104C09 645 141-249 163-173 189-195 228-238 1-123 I104C11 646 141-249 163-173 189-195 228-238 1-123 I104D01 647 141-249 163-173 189-195 228-238 1-123 I104D02 648 141-249 163-173 189-195 228-238 1-123 I104D03 649 141-249 163-173 189-195 228-238 1-123 I104D04 650 141-249 163-173 189-195 228-238 1-123 I104D07 651 141-249 163-173 189-195 228-238 1-123 I104D08 652 141-249 163-173 189-195 228-238 1-123 I104D09 653 141-249 163-173 189-195 228-238 1-123 I104E01 654 141-249 163-173 189-195 228-238 1-123 I104E02 655 141-249 163-173 189-195 228-238 1-123 I104E03 656 141-249 163-173 189-195 228-238 1-123 I104E05 657 141-249 163-173 189-195 228-238 1-123 I104E11 658 141-249 163-173 189-195 228-238 1-123 I104E12 659 141-249 163-173 189-195 228-238 1-123 I104F02 660 141-249 163-173 189-195 228-238 1-123 I104F03 661 141-249 163-173 189-195 228-238 1-123 I104F04 662 141-249 163-173 189-195 228-238 1-123 I104F05 663 141-249 163-173 189-195 228-238 1-123 I104F06 664 141-249 163-173 189-195 228-238 1-123 I104F07 665 141-249 163-173 189-195 228-238 1-123 I104F10 666 141-249 163-173 189-195 228-238 1-123 I104F11 667 141-249 163-173 189-195 228-238 1-123 I104F12 668 141-249 163-173 189-195 228-238 1-123 I104G04 669 141-249 163-173 189-195 228-238 1-123 I104G05 670 141-249 163-173 189-195 228-238 1-123 I104G09 671 141-249 163-173 189-195 228-238 1-123 I104G11 672 141-249 163-173 189-195 228-238 1-123 I105A02 673 141-249 163-173 189-195 228-238 1-123 I105A03 674 141-249 163-173 189-195 228-238 1-123 I105A04 675 141-249 163-173 189-195 228-238 1-123 I105A08 676 141-249 163-173 189-195 228-238 1-123 I105A09 677 141-249 163-173 189-195 228-238 1-123 I105A11 678 141-249 163-173 189-195 228-238 1-123 I105B04 679 141-249 163-173 189-195 228-238 1-123 I105B05 680 141-249 163-173 189-195 228-238 1-123 I105B07 681 141-249 163-173 189-195 228-238 1-123 I105B08 682 141-249 163-173 189-195 228-238 1-123 I105B10 683 141-249 163-173 189-195 228-238 1-123 I105B11 684 141-249 163-173 189-195 228-238 1-123 I105B12 685 141-249 163-173 189-195 228-238 1-123 I105C02 686 141-249 163-173 189-195 228-238 1-123 I105C03 687 141-249 163-173 189-195 228-238 1-123 I105C05 688 141-249 163-173 189-195 228-238 1-123 I105C06 689 141-249 163-173 189-195 228-238 1-123 I105C08 690 141-249 163-173 189-195 228-238 1-123 I105C12 691 141-249 163-173 189-195 228-238 1-123 I105D04 692 141-249 163-173 189-195 228-238 1-123 I105D06 693 141-249 163-173 189-195 228-238 1-123 I105D08 694 141-249 163-173 189-195 228-238 1-123 I105D09 695 141-249 163-173 189-195 228-238 1-123 I105D10 696 141-249 163-173 189-195 228-238 1-123 I105D11 697 141-249 163-173 189-195 228-238 1-123 I105E01 698 141-249 163-173 189-195 228-238 1-123 I105E06 699 141-249 163-173 189-195 228-238 1-123 I105E11 700 141-249 163-173 189-195 228-238 1-123 I105F03 701 141-249 163-173 189-195 228-238 1-123 I105F06 702 141-249 163-173 189-195 228-238 1-123 I105F07 703 141-249 163-173 189-195 228-238 1-123 I105F09 704 141-249 163-173 189-195 228-238 1-123 I105F12 705 141-249 163-173 189-195 228-238 1-123 I105G03 706 141-249 163-173 189-195 228-238 1-123 I105G08 707 141-249 163-173 189-195 228-238 1-123 I105G09 708 141-249 163-173 189-195 228-238 1-123 I105G10 709 141-249 163-173 189-195 228-238 1-123 I105G11 710 141-249 163-173 189-195 228-238 1-123 I107A01 711 141-249 163-173 189-195 228-238 1-123 I107A03 712 141-249 163-173 189-195 228-238 1-123 I107A06 713 141-249 163-173 189-195 228-238 1-123 I107A07 714 141-249 163-173 189-195 228-238 1-123 I107A09 715 141-249 163-173 189-195 228-238 1-123 I107A12 716 141-249 163-173 189-195 228-238 1-123 I107B02 717 141-249 163-173 189-195 228-238 1-123 I107B04 718 141-249 163-173 189-195 228-238 1-123 I107B05 719 141-249 163-173 189-195 228-238 1-123 I107C01 720 139-247 161-171 187-193 226-236 1-121 I107C02 721 141-249 163-173 189-195 228-238 1-123 I107C04 722 141-249 163-173 189-195 228-238 1-123 I107C06 723 141-249 163-173 189-195 228-238 1-123 I107C08 724 141-249 163-173 189-195 228-238 1-123 I107C10 725 141-249 163-173 189-195 228-238 1-123 I107D01 726 141-249 163-173 189-195 228-238 1-123 I107D04 727 141-249 163-173 189-195 228-238 1-123 I107D07 728 141-249 163-173 189-195 228-238 1-123 I107D12 729 141-249 163-173 189-195 228-238 1-123 I107E01 730 141-249 163-173 189-195 228-238 1-123 I107E05 731 141-249 163-173 189-195 228-238 1-123 I107E07 732 141-249 163-173 189-195 228-238 1-123 I107E09 733 141-249 163-173 189-195 228-238 1-123 I107F01 734 141-249 163-173 189-195 228-238 1-123 I107F05 735 141-249 163-173 189-195 228-238 1-123 I107F09 736 141-249 163-173 189-195 228-238 1-123 I107F10 737 141-249 163-173 189-195 228-238 1-123 I107G01 738 141-249 163-173 189-195 228-238 1-123 I107G05 739 141-249 163-173 189-195 228-238 1-123 I107H02 740 141-249 163-173 189-195 228-238 1-123 I107H06 741 141-249 163-173 189-195 228-238 1-123 I107H09 742 141-249 163-173 189-195 228-238 1-123 I107H10 743 141-249 163-173 189-195 228-238 1-123 I108A12 744 141-249 163-173 189-195 228-238 1-123 I108B03 745 141-249 163-173 189-195 228-238 1-123 I108B04 746 141-249 163-173 189-195 228-238 1-123 I108C09 747 141-249 163-173 189-195 228-238 1-123 I108C11 748 141-249 163-173 189-195 228-238 1-123 I108D10 749 141-249 163-173 189-195 228-238 1-123 I108D11 750 141-249 163-173 189-195 228-238 1-123 I108D12 751 141-249 163-173 189-195 228-238 1-123 I108E01 752 141-249 163-173 189-195 228-238 1-123 I108E03 753 141-249 163-173 189-195 228-238 1-123 I108E05 754 141-249 163-173 189-195 228-238 1-123 I108E07 755 141-249 163-173 189-195 228-238 1-123 I108E08 756 141-249 163-173 189-195 228-238 1-123 I108E09 757 141-249 163-173 189-195 228-238 1-123 I108E10 758 141-249 163-173 189-195 228-238 1-123 I108E11 759 141-249 163-173 189-195 228-238 1-123 I108F10 760 141-249 163-173 189-195 228-238 1-123 I108F12 761 141-249 163-173 189-195 228-238 1-123 I108G01 762 141-249 163-173 189-195 228-238 1-123 I108G02 763 141-249 163-173 189-195 228-238 1-123 I108G07 764 141-249 163-173 189-195 228-238 1-123 I108G10 765 141-249 163-173 189-195 228-238 1-123 I108G11 766 141-249 163-173 189-195 228-238 1-123 I108G12 767 141-249 163-173 189-195 228-238 1-123 I108H01 768 141-249 163-173 189-195 228-238 1-123 I108H02 769 141-249 163-173 189-195 228-238 1-123 I108H06 770 141-249 163-173 189-195 228-238 1-123 I108H08 771 141-249 163-173 189-195 228-238 1-123 I111A06 772 141-249 163-173 189-195 228-238 1-123 I111B12 773 141-249 163-173 189-195 228-238 1-123 I111C01 774 141-249 163-173 189-195 228-238 1-123 I111D06 775 141-249 163-173 189-195 228-238 1-123 I111E04 776 141-249 163-173 189-195 228-238 1-123 I111E10 777 141-249 163-173 189-195 228-238 1-123 I111E11 778 141-250 163-173 189-195 229-239 1-123 I111E12 779 141-249 163-173 189-195 228-238 1-123 I111F07 780 141-249 163-173 189-195 228-238 1-123 I111G02 781 141-249 163-173 189-195 228-238 1-123 I111H10 782 141-249 163-173 189-195 228-238 1-123 I113A04 783 141-249 163-173 189-195 228-238 1-123 I113A12 784 141-249 163-173 189-195 228-238 1-123 I113B06 785 141-249 163-173 189-195 228-238 1-123 I113C06 786 141-249 163-173 189-195 228-238 1-123 I113G04 787 141-249 163-173 189-195 228-238 1-123 I113G05 788 141-249 163-173 189-195 228-238 1-123 I113G10 789 141-249 163-173 189-195 228-238 1-123 I113G11 790 141-249 163-173 189-195 228-238 1-123 I113H06 791 141-249 163-173 189-195 228-238 1-123 I113H07 792 141-249 163-173 189-195 228-238 1-123 I113H09 793 141-249 163-173 189-195 228-238 1-123 I114C04 794 141-249 163-173 189-195 228-238 1-123 I114C12 795 141-249 163-173 189-195 228-238 1-123 I114D04 796 141-249 163-173 189-195 228-238 1-123 I114D06 797 141-249 163-173 189-195 228-238 1-123 I114D10 798 141-249 163-173 189-195 228-238 1-123 I114E01 799 141-249 163-173 189-195 228-238 1-123 I114E02 800 141-249 163-173 189-195 228-238 1-123 I114E03 801 141-249 163-173 189-195 228-238 1-123 I114E11 802 141-249 163-173 189-195 228-238 1-123 I114H01 803 141-249 163-173 189-195 228-238 1-123 I114H06 804 141-249 163-173 189-195 228-238 1-123 I114H09 805 141-249 163-173 189-195 228-238 1-123 I115A02 806 141-249 163-173 189-195 228-238 1-123 I115A07 807 141-249 163-173 189-195 228-238 1-123 I115B10 808 141-249 163-173 189-195 228-238 1-123 I115C05 809 141-249 163-173 189-195 228-238 1-123 I115C06 810 141-249 163-173 189-195 228-238 1-123 I115C08 811 141-249 163-173 189-195 228-238 1-123 I115C12 812 141-249 163-173 189-195 228-238 1-123 I115D07 813 141-249 163-173 189-195 228-238 1-123 I115E09 814 141-249 163-173 189-195 228-238 1-123 I115F06 815 141-249 163-173 189-195 228-238 1-123 I115F07 816 141-249 163-173 189-195 228-238 1-123 I115F12 817 141-249 163-173 189-195 228-238 1-123 I115G04 818 141-249 163-173 189-195 228-238 1-123 I115G05 819 141-249 163-173 189-195 228-238 1-123 I115G08 820 141-249 163-173 189-195 228-238 1-123 I115H04 821 141-249 163-173 189-195 228-238 1-123 I115H07 822 141-249 163-173 189-195 228-238 1-123 I115H09 823 141-249 163-173 189-195 228-238 1-123 I116A07 824 141-249 163-173 189-195 228-238 1-123 I116B01 825 141-249 163-173 189-195 228-238 1-123 I116B12 826 141-249 163-173 189-195 228-238 1-123 I116C06 827 141-249 163-173 189-195 228-238 1-123 I116D07 828 141-249 163-173 189-195 228-238 1-123 I116E02 829 141-249 163-173 189-195 228-238 1-123 I116E04 830 141-249 163-173 189-195 228-238 1-123 I116F02 831 141-249 163-173 189-195 228-238 1-123 I116F11 832 141-249 163-173 189-195 228-238 1-123 I116G05 833 141-249 163-173 189-195 228-238 1-123 I001C09 834 143-250 164-174 190-196 229-239 1-127 I006D07 835 141-248 162-172 188-194 227-237 1-125 I007B03 836 143-253 165-178 194-200 233-242 1-127 I007F11 837 140-250 162-175 191-197 230-239 1-124 I007H08 838 144-254 166-179 195-201 234-243 1-128 I008A09 839 146-256 168-181 197-203 236-245 1-130 I008B01 840 141-251 163-176 192-198 231-240 1-125 I008C02 841 145-255 167-180 196-202 235-244 1-129 I008C03 842 143-250 164-174 190-196 229-239 1-127 I008C12 843 146-256 168-181 197-203 236-245 1-130 I012A06 844 147-254 169-179 195-201 234-243 1-129 I016E05 845 141-251 163-176 192-198 231-240 1-125 I016F02 846 135-245 157-170 186-192 225-234 1-119 I016F04 847 141-251 163-176 192-198 231-240 1-125 I016H07 848 141-248 162-172 188-194 227-237 1-125 I018C02 849 141-251 163-176 192-198 231-240 1-125 I018C10 850 143-250 164-174 190-196 229-239 1-127 I018D07 851 143-250 164-174 190-196 229-239 1-127 I018H08 852 141-251 163-176 192-198 231-240 1-125 I018H09 853 141-251 163-176 192-198 231-240 1-125 I021B05 854 143-253 165-178 194-200 233-242 1-127 I022E02 855 141-251 163-176 192-198 231-240 1-125 I026E03 856 143-251 165-175 191-197 230-240 1-125 I027A07 857 145-255 167-179 195-201 234-244 1-128 I028A06 858 142-253 164-176 192-198 231-242 1-126 I029D07 859 141-250 163-176 192-198 231-239 1-125 I029F11 860 143-253 165-177 193-199 232-242 1-127 I031C03 861 138-248 160-172 188-194 227-237 1-121 I031C07 862 148-258 170-183 199-205 238-247 1-131 I031F09 863 145-255 167-179 195-201 234-244 1-127 I031G08 864 148-259 170-182 198-204 237-248 1-131 I031G10 865 148-258 170-182 198-204 237-247 1-131 I031G11 866 145-255 167-179 195-201 234-244 1-127 I037E07 867 140-250 162-175 191-197 230-239 1-124 I037E12 868 140-250 162-175 191-197 230-239 1-124 I050A07 869 146-257 168-181 197-203 236-246 1-129 I061D02 870 144-254 166-179 195-201 234-243 1-128 I061E07 871 141-251 163-175 191-197 230-240 1-125 I061H01 872 146-256 168-181 197-203 236-245 1-130 I001A03 873 144-254 166-179 195-201 234-243 1-128 I001A07 874 141-251 163-176 192-198 231-240 1-125 I001A08 875 141-251 163-176 192-198 231-240 1-125 I001A10 876 141-251 163-176 192-198 231-240 1-125 I001A12 877 141-248 162-172 188-194 227-237 1-125 I001B02 878 137-247 159-171 187-193 226-236 1-121 I001B07 879 141-251 163-176 192-198 231-240 1-125 I001C06 880 143-253 165-178 194-200 233-242 1-127 I001C08 881 144-254 166-179 195-201 234-243 1-128 I001C12 882 141-251 163-176 192-198 231-240 1-125 I001D08 883 140-250 162-175 191-197 230-239 1-124 I001D12 884 141-251 163-176 192-198 231-240 1-125 I001E05 885 143-253 165-178 194-200 233-242 1-127 I001E07 886 141-251 163-176 192-198 231-240 1-125 I001G09 887 141-251 163-176 192-198 231-240 1-125 I001H05 888 144-254 166-179 195-201 234-243 1-128 I001H08 889 141-251 163-176 192-198 231-240 1-125 I003A01 890 141-251 163-176 192-198 231-240 1-124 I003A06 891 141-251 163-176 192-198 231-240 1-124 I003A07 892 142-249 163-173 189-195 228-238 1-126 I003A10 893 141-248 162-172 188-194 227-237 1-125 I003B03 894 141-251 163-176 192-198 231-240 1-124 I003B04 895 140-248 162-172 188-194 227-237 1-122 I003B09 896 142-249 163-173 189-195 228-238 1-126 I003C01 897 142-252 164-176 192-198 231-241 1-124 I003C02 898 142-252 164-176 192-198 231-241 1-125 I003C03 899 142-250 164-174 190-196 229-239 1-125 I003C12 900 141-248 162-172 188-194 227-237 1-125 I003D04 901 140-250 162-174 190-196 229-239 1-123 I003E05 902 142-253 164-176 192-198 231-242 1-125 I003F01 903 141-251 163-176 192-198 231-240 1-124 I003F02 904 140-251 162-175 191-197 230-240 1-123 I003G01 905 145-254 168-179 195-201 234-243 1-127 I003G05 906 144-255 166-179 195-201 234-244 1-127 I003G06 907 146-256 168-181 197-203 236-245 1-129 I003G11 908 144-251 165-175 191-197 230-240 1-128 I003H02 909 142-253 164-176 192-198 233-242 1-124 I003H05 910 141-251 163-176 192-198 231-240 1-124 I003H08 911 142-249 163-173 189-195 228-238 1-126 I005A01 912 141-249 162-172 188-194 227-238 1-125 I005A02 913 141-248 162-172 188-194 227-237 1-125 I005B01 914 141-248 162-172 188-194 227-237 1-125 I005B09 915 137-247 159-172 188-194 227-236 1-121 I005C01 916 141-248 162-172 188-194 227-237 1-125 I005D02 917 141-251 163-176 192-198 231-240 1-125 I005D03 918 142-249 165-175 191-197 230-238 1-126 I005E01 919 142-249 165-175 191-197 230-238 1-126 I005E08 920 141-248 162-172 188-194 227-237 1-125 I005F01 921 142-248 164-174 190-196 229-238 1-124 I005F02 922 144-251 167-177 193-199 232-240 1-128 I005F04 923 137-247 159-172 188-194 227-236 1-121 I005F08 924 140-247 161-171 187-193 226-236 1-124 I005G01 925 141-251 163-176 192-198 231-240 1-125 I005G08 926 142-249 165-175 191-197 230-238 1-126 I005H02 927 140-247 161-171 187-193 226-236 1-124 I006B01 928 139-246 160-170 186-192 225-235 1-123 I006C09 929 143-253 165-177 193-199 232-242 1-127 I006D09 930 141-251 163-176 192-198 231-240 1-125 I006E01 931 143-253 165-178 194-200 233-242 1-127 I006E07 932 143-250 166-176 192-198 231-239 1-127 I006F01 933 140-250 162-175 191-197 230-239 1-124 I006F02 934 142-253 164-176 192-198 231-242 1-126 I006F07 935 143-253 165-177 193-199 232-242 1-127 I006G01 936 146-253 169-179 195-201 234-242 1-130 I006G04 937 132-239 153-163 179-185 218-228 1-116 I006H01 938 146-253 167-177 193-199 232-242 1-130 I006H02 939 143-253 165-177 193-199 232-242 1-127 I007A01 940 141-251 163-176 192-198 231-240 1-125 I007A08 941 139-249 161-174 190-196 229-238 1-123 I007A11 942 140-250 162-175 191-197 230-239 1-124 I007A12 943 144-251 165-175 191-197 230-240 1-128 I007B04 944 141-251 163-176 192-198 231-240 1-125 I007C04 945 141-251 163-176 192-198 231-240 1-125 I007C08 946 142-249 163-173 189-195 228-238 1-126 I007C12 947 140-250 162-175 191-197 230-239 1-124 I007D07 948 140-247 161-171 187-193 226-236 1-124 I007D08 949 144-251 165-175 191-197 230-240 1-128 I007E03 950 141-248 162-172 188-194 227-237 1-125 I007E10 951 141-251 163-176 192-198 231-240 1-125 I007E11 952 144-251 165-175 191-197 230-240 1-128 I007F06 953 141-248 162-172 188-194 227-237 1-125 I007F08 954 143-253 165-178 194-200 233-242 1-127 I007G07 955 141-251 163-176 192-198 231-240 1-125 I007G09 956 142-252 164-177 193-199 232-241 1-126 I007G10 957 142-249 163-173 189-195 228-238 1-126 I007H07 958 147-257 169-182 198-204 237-246 1-131 I007H11 959 141-248 162-172 188-194 227-237 1-125 I008A02 960 141-251 163-176 192-198 231-240 1-125 I008A05 961 141-251 163-176 192-198 231-240 1-125 I008A06 962 141-251 163-176 192-198 231-240 1-125 I008A07 963 142-249 163-173 189-195 228-238 1-126 I008A12 964 140-250 162-175 191-197 230-239 1-124 I008B02 965 141-248 162-172 188-194 227-237 1-125 I008B04 966 143-253 165-178 194-200 233-242 1-127 I008B05 967 141-248 162-172 188-194 227-237 1-125 I008B06 968 141-251 163-176 192-198 231-240 1-125 I008B07 969 140-247 163-173 189-195 228-236 1-124 I008B10 970 141-248 162-172 188-194 227-237 1-125 I008B11 971 144-254 166-179 195-201 234-243 1-128 I008C06 972 141-251 163-176 192-198 231-240 1-125 I008C08 973 149-259 171-183 199-205 238-248 1-133 I008C09 974 142-249 163-173 189-195 228-238 1-126 I008D01 975 141-251 163-176 192-198 231-240 1-125 I008D02 976 141-251 163-176 192-198 231-240 1-125 I008D03 977 144-254 166-179 195-201 234-243 1-128 I008D04 978 141-251 163-176 192-198 231-240 1-125 I008D05 979 141-251 163-176 192-198 231-240 1-125 I008D06 980 141-248 162-172 188-194 227-237 1-125 I008D07 981 144-254 166-179 195-201 234-243 1-128 I008D08 982 144-251 165-175 191-197 230-240 1-128 I008D12 983 144-254 166-179 195-201 234-243 1-128 I008E01 984 141-248 162-172 188-194 227-237 1-125 I008E02 985 137-247 159-172 188-194 227-236 1-121 I008E03 986 141-251 163-176 192-198 231-240 1-125 I008E04 987 141-248 162-172 188-194 227-237 1-125 I008E08 988 141-252 163-175 191-197 230-241 1-125 I008E09 989 143-253 165-178 194-200 233-242 1-127 I008E12 990 141-251 163-176 192-198 231-240 1-125 I008F03 991 141-251 163-176 192-198 231-240 1-125 I008F06 992 141-251 163-176 192-198 231-240 1-125 I008F07 993 143-250 164-174 190-196 229-239 1-127 I008F08 994 143-253 165-178 194-200 233-242 1-127 I008F09 995 133-243 155-168 184-190 223-232 1-117 I008F10 996 140-247 161-171 187-193 226-236 1-124 I008F11 997 144-251 165-175 191-197 230-240 1-128 I008G02 998 141-251 163-176 192-198 231-240 1-125 I008G03 999 140-247 161-171 187-193 226-236 1-124 I008G04 1000 143-253 165-178 194-200 233-242 1-127 I008G05 1001 144-254 166-179 195-201 234-243 1-128 I008G11 1002 136-246 158-171 187-193 226-235 1-120 I008G12 1003 143-253 165-178 194-200 233-242 1-127 I008H02 1004 141-248 164-174 190-196 229-237 1-125 I008H03 1005 141-251 163-176 192-198 231-240 1-125 I008H06 1006 143-253 165-178 194-200 233-242 1-127 I008H09 1007 143-253 165-178 194-200 233-242 1-127 I008H11 1008 141-248 164-174 190-196 229-237 1-125 I012B03 1009 141-249 163-175 191-197 230-238 1-124 I012B06 1010 141-251 163-176 192-198 231-240 1-124 I012B10 1011 141-251 163-175 191-197 230-240 1-124 I012C03 1012 143-255 165-178 194-200 233-244 1-126 I012C06 1013 141-251 163-176 192-198 231-240 1-124 I012C09 1014 142-250 164-174 190-196 229-239 1-124 I012D12 1015 146-256 168-180 196-202 235-245 1-129 I012E07 1016 142-252 164-176 192-198 231-241 1-124 I012E08 1017 140-250 162-174 190-196 229-239 1-123 I012E09 1018 141-247 163-173 189-195 228-236 1-124 I012F05 1019 141-249 163-173 189-195 228-238 1-124 I012F12 1020 142-251 164-176 192-198 231-240 1-124 I012G03 1021 142-252 164-176 192-198 231-241 1-124 I012G05 1022 141-250 163-173 189-195 228-239 1-123 I012G10 1023 140-251 162-175 191-197 230-240 1-123 I012H09 1024 141-249 163-173 189-195 228-238 1-124 I013A10 1025 148-259 170-182 198-204 237-248 1-131 I013A12 1026 149-256 171-181 197-203 236-245 1-131 I013B04 1027 149-256 172-182 198-204 237-245 1-131 I013B09 1028 149-257 171-181 197-203 236-246 1-131 I013C02 1029 148-258 170-182 198-204 237-247 1-131 I013C04 1030 139-249 161-173 189-195 228-238 1-121 I013D02 1031 138-248 160-173 189-195 228-237 1-121 I013D03 1032 148-259 170-183 199-205 238-248 1-131 I013D10 1033 146-257 168-181 197-203 236-246 1-129 I013E02 1034 148-259 170-183 199-205 238-248 1-131 I013E05 1035 139-249 162-173 189-195 228-238 1-121 I013E09 1036 148-260 170-183 199-205 238-249 1-131 I013F03 1037 138-248 160-172 188-194 227-237 1-121 I013F04 1038 148-258 170-182 198-204 237-247 1-131 I013F07 1039 147-260 170-185 201-207 240-249 1-129 I013F09 1040 138-248 160-172 188-194 227-237 1-121 I013F10 1041 148-259 170-183 199-205 238-248 1-131 I013H04 1042 148-258 170-182 198-204 237-247 1-131 I013H07 1043 148-259 170-183 199-205 238-248 1-131 I014A12 1044 143-253 165-178 194-200 233-242 1-127 I014C06 1045 142-254 164-177 193-200 233-243 1-125 I014C10 1046 141-251 163-176 192-198 231-240 1-125 I014C12 1047 141-251 163-176 192-198 231-240 1-125 I014E06 1048 142-252 164-176 192-198 231-241 1-124 I014F02 1049 143-251 166-176 192-198 231-240 1-125 I016A08 1050 144-251 165-175 191-197 230-240 1-128 I016A09 1051 141-251 163-176 192-198 231-240 1-125 I016C02 1052 141-251 163-176 192-198 231-240 1-125 I016C03 1053 141-251 163-176 192-198 231-240 1-125 I016C05 1054 148-255 169-179 195-201 234-244 1-132 I016C09 1055 141-251 163-176 192-198 231-240 1-125 I016C11 1056 148-255 169-179 195-201 234-244 1-132 I016D10 1057 141-251 163-176 192-198 231-240 1-125 I016D11 1058 141-251 163-176 192-198 231-240 1-125 I016E03 1059 141-251 163-176 192-198 231-240 1-125 I016E04 1060 141-251 163-176 192-198 231-240 1-125 I016F03 1061 141-251 163-176 192-198 231-240 1-125 I016F11 1062 141-251 163-176 192-198 231-240 1-125 I016G01 1063 141-251 163-176 192-198 231-240 1-125 I016G06 1064 141-251 163-176 192-198 231-240 1-125 I016G12 1065 148-255 169-179 195-201 234-244 1-132 I016H10 1066 141-251 163-176 192-198 231-240 1-125 I017A06 1067 141-251 163-176 192-198 231-240 1-125 I017A07 1068 141-251 163-176 192-198 231-240 1-125 I017A11 1069 140-253 162-175 191-197 233-242 1-124 I017E12 1070 141-251 163-176 192-198 231-240 1-125 I017G03 1071 141-251 163-176 192-198 231-240 1-125 I017G07 1072 141-251 163-176 192-198 231-240 1-125 I017H01 1073 141-251 163-176 192-198 231-240 1-125 I018A02 1074 141-251 163-176 192-198 231-240 1-125 I018A04 1075 144-254 166-179 195-201 234-243 1-128 I018A05 1076 141-251 163-176 192-198 231-240 1-125 I018A11 1077 141-251 163-176 192-198 231-240 1-125 I018B02 1078 141-251 163-176 192-198 231-240 1-125 I018B08 1079 141-251 163-176 192-198 231-240 1-125 I018C04 1080 141-251 163-176 192-198 231-240 1-125 I018D02 1081 141-251 163-176 192-198 231-240 1-125 I018E06 1082 141-251 163-176 192-198 231-240 1-125 I018E08 1083 141-251 163-176 192-198 231-240 1-125 I018F04 1084 141-251 163-176 192-198 231-240 1-125 I018G06 1085 141-251 163-176 192-198 231-240 1-125 I018H07 1086 141-251 163-176 192-198 231-240 1-125 I019E05 1087 144-254 166-179 195-201 234-243 1-128 I019F06 1088 144-254 166-179 195-201 234-243 1-128 I019G12 1089 143-253 165-178 194-200 233-242 1-127 I020D01 1090 137-247 159-171 187-193 226-236 1-121 I020D05 1091 143-253 165-178 194-200 233-242 1-127 I020E10 1092 143-253 165-178 194-200 233-242 1-127 I020G12 1093 143-253 165-178 194-200 233-242 1-127 I020H06 1094 143-253 165-178 194-200 233-242 1-127 I020H10 1095 143-253 165-178 194-200 233-242 1-127 I021A11 1096 143-253 165-178 194-200 233-242 1-127 I021B01 1097 143-253 165-178 194-200 233-242 1-127 I021C11 1098 143-253 165-178 194-200 233-242 1-127 I021D12 1099 137-247 159-171 187-193 226-236 1-121 I021E10 1100 143-253 165-178 194-200 233-242 1-127 I021G02 1101 143-253 165-178 194-200 233-242 1-127 I022A08 1102 142-249 163-173 189-195 228-238 1-126 I022B01 1103 141-251 163-176 192-198 231-240 1-125 I022B10 1104 141-248 164-174 190-196 229-237 1-125 I022C02 1105 142-249 163-173 189-195 228-238 1-126 I022C04 1106 141-251 163-176 192-198 231-240 1-125 I022C08 1107 141-251 163-176 192-198 231-240 1-125 I022D06 1108 142-249 163-173 189-195 228-238 1-126 I022E08 1109 142-249 163-173 189-195 228-238 1-126 I022F01 1110 142-249 163-173 189-195 228-238 1-126 I022F04 1111 142-249 163-173 189-195 228-238 1-126 I022F12 1112 140-247 161-171 187-193 226-236 1-124 I022G11 1113 142-249 163-173 189-195 228-238 1-126 I023D01 1114 141-251 163-176 192-198 231-240 1-125 I023D04 1115 142-249 163-173 189-195 228-238 1-126 I024B04 1116 140-247 161-171 187-193 226-236 1-124 I024D01 1117 142-249 163-173 189-195 228-238 1-126 I024F06 1118 142-249 163-173 189-195 228-238 1-126 I024H01 1119 142-249 163-173 189-195 228-238 1-126 I024H07 1120 142-249 163-173 189-195 228-238 1-126 I025A01 1121 141-251 163-176 192-198 231-240 1-124 I025A04 1122 141-251 163-175 191-197 230-240 1-124 I025A07 1123 141-249 163-173 189-195 228-238 1-124 I025B01 1124 134-244 156-168 184-190 223-233 1-117 I025B10 1125 142-253 164-176 192-198 233-242 1-124 I025B12 1126 141-251 163-176 192-198 231-240 1-124 I025C07 1127 141-251 163-176 192-198 231-240 1-124 I025D11 1128 142-252 164-176 192-198 231-241 1-124 I025E04 1129 142-252 164-176 192-198 231-241 1-126 I025E05 1130 141-251 163-175 191-197 230-240 1-124 I025E07 1131 142-252 164-176 192-198 231-241 1-124 I025E10 1132 141-251 163-176 192-198 231-240 1-125 I025F01 1133 140-251 162-175 191-197 230-240 1-123 I025F08 1134 138-248 160-172 188-194 227-237 1-121 I025G03 1135 142-252 164-176 192-198 231-241 1-124 I025G08 1136 141-254 163-176 192-198 231-243 1-124 I025H02 1137 145-255 167-179 195-201 234-244 1-128 I026A01 1138 143-249 165-175 191-197 230-238 1-125 I026B01 1139 144-254 166-178 194-200 233-243 1-127 I026B06 1140 141-251 163-176 192-198 231-240 1-124 I026C06 1141 141-251 163-176 192-198 231-240 1-124 I026C10 1142 139-249 161-174 190-196 229-238 1-122 I026C11 1143 141-251 163-176 192-198 231-240 1-125 I026D09 1144 140-252 162-175 191-197 230-241 1-123 I026E04 1145 142-252 164-176 192-198 231-241 1-124 I026E06 1146 141-251 163-175 191-197 230-240 1-124 I026E09 1147 141-251 163-176 192-198 231-240 1-124 I026F01 1148 141-251 163-176 192-198 231-240 1-124 I026F09 1149 141-251 163-176 192-198 231-240 1-124 I026F12 1150 141-256 163-176 192-202 237-245 1-124 I026G08 1151 141-251 163-176 192-198 231-240 1-124 I026G10 1152 141-251 163-176 192-198 231-240 1-124 I026G11 1153 144-255 166-179 195-201 234-244 1-127 I026H02 1154 140-251 162-175 191-197 230-240 1-123 I026H06 1155 141-251 163-175 191-197 230-240 1-124 I026H10 1156 145-255 167-179 195-201 234-244 1-128 I027A09 1157 141-251 163-176 192-198 231-240 1-124 I027B02 1158 140-250 162-174 190-196 229-239 1-123 I027B05 1159 141-250 163-176 192-198 230-239 1-124 I027C08 1160 139-249 161-174 190-196 229-238 1-122 I027D02 1161 142-250 164-174 190-196 229-239 1-125 I027E03 1162 141-251 163-176 192-198 231-240 1-124 I027E05 1163 142-252 164-176 192-198 231-241 1-124 I027F04 1164 145-252 167-176 192-198 231-241 1-128 I027F05 1165 141-254 163-176 192-198 231-243 1-124 I027F11 1166 141-251 163-176 192-198 231-240 1-124 I027G06 1167 142-253 164-176 192-198 233-242 1-124 I027G07 1168 142-250 164-174 190-196 229-239 1-124 I027H03 1169 142-252 164-176 192-198 231-241 1-125 I028A04 1170 143-250 164-174 190-196 229-239 1-127 I028A07 1171 141-251 163-176 192-198 231-240 1-125 I028B08 1172 141-251 163-176 192-198 231-240 1-125 I028B10 1173 148-258 170-183 199-205 238-247 1-132 I028C01 1174 142-250 165-175 191-197 230-239 1-126 I028C04 1175 143-253 165-178 194-200 233-242 1-127 I028C08 1176 141-251 163-176 192-198 231-240 1-125 I028D04 1177 140-247 163-173 189-195 228-236 1-124 I028D05 1178 141-248 162-172 188-194 227-237 1-125 I028D12 1179 143-250 164-174 190-196 229-239 1-127 I028E06 1180 143-253 165-178 194-200 233-242 1-127 I028E07 1181 141-248 162-172 188-194 227-237 1-125 I028E08 1182 141-248 162-172 188-194 227-237 1-125 I028F06 1183 146-256 168-180 196-202 235-245 1-130 I028F08 1184 134-244 156-169 185-191 224-233 1-118 I028G08 1185 141-251 163-176 192-198 231-240 1-125 I028G09 1186 144-254 166-179 195-201 234-243 1-128 I028G10 1187 141-251 163-176 192-198 231-240 1-125 I028H02 1188 142-249 165-175 191-197 230-238 1-126 I028H03 1189 148-256 169-179 195-201 234-245 1-132 I028H06 1190 145-255 167-180 196-202 235-244 1-129 I028H09 1191 140-250 162-175 191-197 230-239 1-124 I029A10 1192 139-246 160-170 186-192 225-235 1-123 I029A12 1193 137-247 159-171 187-193 226-236 1-121 I029B11 1194 141-251 163-176 192-198 231-240 1-125 I029C08 1195 144-254 166-179 195-201 234-243 1-128 I029E10 1196 144-254 166-179 195-201 234-243 1-128 I029F08 1197 144-254 166-179 195-201 234-243 1-128 I029G08 1198 141-248 162-172 188-194 227-237 1-125 I030A02 1199 143-253 165-177 193-199 232-242 1-126 I030A03 1200 141-253 163-175 191-197 230-242 1-124 I030A04 1201 141-252 163-176 192-198 231-241 1-124 I030A05 1202 141-249 163-175 191-197 230-238 1-124 I030A09 1203 140-250 162-174 190-196 229-239 1-123 I030A12 1204 141-249 163-173 189-195 228-238 1-123 I030B06 1205 141-249 163-173 189-195 228-238 1-123 I030B08 1206 141-247 163-173 189-195 228-236 1-124 I030B10 1207 143-251 165-175 191-197 230-240 1-125 I0300O3 1208 140-252 162-175 191-197 230-241 1-123 I030006 1209 147-256 169-182 198-204 237-245 1-130 I030008 1210 134-244 156-168 184-190 223-233 1-117 I030009 1211 141-251 163-175 191-197 230-240 1-124 I030C10 1212 141-250 163-175 191-197 230-239 1-124 I030C11 1213 140-251 162-175 191-197 230-240 1-123 I030C12 1214 134-244 156-168 184-190 223-233 1-117 I030D07 1215 141-249 163-173 189-195 228-238 1-123 I030D12 1216 141-251 163-175 191-197 230-240 1-124 I030E02 1217 140-251 162-175 191-197 230-240 1-123 I030E05 1218 142-252 164-176 192-198 231-241 1-124 I030E07 1219 142-251 165-176 192-198 231-240 1-124 I030E08 1220 141-251 163-175 191-197 230-240 1-124 I030E09 1221 141-252 163-176 192-198 231-241 1-124 I030E10 1222 140-250 162-174 190-196 229-239 1-123 I030F02 1223 142-252 164-176 192-198 231-241 1-125 I030F05 1224 141-251 163-175 191-197 230-240 1-124 I030F06 1225 140-251 162-175 191-197 230-240 1-123 I030F08 1226 141-254 163-176 192-198 231-243 1-124 I030F09 1227 142-253 164-176 192-198 231-242 1-124 I030F11 1228 140-250 162-174 190-196 229-239 1-123 I030F12 1229 141-251 163-175 191-197 230-240 1-124 I030G03 1230 141-256 163-176 192-202 237-245 1-124 I030G07 1231 140-251 162-175 191-197 230-240 1-123 I030G09 1232 142-251 164-174 190-196 229-240 1-124 I030H05 1233 146-255 168-181 197-203 236-244 1-129 I030H06 1234 148-258 170-182 198-204 239-247 1-130 I030H10 1235 141-253 163-176 192-198 231-242 1-124 I030H11 1236 142-252 164-176 192-198 231-241 1-124 I031A01 1237 138-248 160-173 189-195 228-237 1-121 I031A03 1238 143-251 166-176 192-198 231-240 1-125 I031A08 1239 148-258 170-182 198-204 237-247 1-131 I031A12 1240 147-257 169-181 197-203 236-246 1-130 I031B03 1241 137-246 159-172 188-194 227-235 1-120 I031B06 1242 143-253 165-177 193-199 232-242 1-126 I031B07 1243 148-258 170-182 198-204 237-247 1-131 I031B08 1244 149-260 171-183 199-205 238-249 1-131 I031B09 1245 148-258 170-182 198-204 237-247 1-131 I031B11 1246 138-248 160-172 188-194 227-237 1-121 I031B12 1247 148-259 170-183 199-205 238-248 1-131 I031C01 1248 138-248 160-172 188-194 227-237 1-121 I031C02 1249 142-253 164-177 193-199 232-242 1-125 I031C04 1250 149-260 171-183 199-205 238-249 1-131 I031C08 1251 139-248 161-171 187-193 226-237 1-121 I031C11 1252 149-257 171-181 197-203 236-246 1-131 I031D01 1253 146-256 168-180 196-202 235-245 1-129 I031D04 1254 138-248 160-172 188-194 227-237 1-121 I031D06 1255 148-258 170-182 198-204 237-247 1-131 I031D08 1256 145-257 167-180 196-202 235-246 1-128 I031D09 1257 139-247 161-171 187-193 226-236 1-121 I031D11 1258 149-256 171-181 197-203 236-245 1-131 I031D12 1259 146-254 168-178 194-200 233-243 1-128 I031E01 1260 148-258 170-182 198-204 237-247 1-131 I031E05 1261 149-257 171-181 197-203 236-246 1-131 I031E07 1262 148-259 170-182 198-204 237-248 1-131 I031E08 1263 148-259 170-183 199-205 238-248 1-131 I031E09 1264 139-246 162-173 189-195 228-235 1-121 I031E10 1265 148-258 170-182 198-204 237-247 1-131 I031E11 1266 148-258 170-182 198-204 237-247 1-131 I031F01 1267 138-248 160-172 188-194 227-237 1-121 I031F04 1268 139-246 162-172 188-194 227-235 1-121 I031F06 1269 137-247 159-171 187-193 226-236 1-119 I031F10 1270 148-259 170-183 199-205 238-248 1-131 I031F11 1271 145-255 167-179 195-201 234-244 1-128 I031F12 1272 138-249 160-172 188-194 227-238 1-121 I031G01 1273 138-248 160-172 188-194 227-237 1-121 I031G03 1274 148-258 170-182 198-204 237-247 1-131 I031G05 1275 148-259 170-183 199-205 238-248 1-131 I031G06 1276 148-258 170-182 198-204 237-247 1-131 I031G07 1277 149-259 171-183 199-205 238-248 1-131 I031G09 1278 148-263 170-183 199-209 244-252 1-131 I031G12 1279 146-256 168-180 196-202 235-245 1-129 I031H01 1280 138-250 160-173 189-195 228-239 1-121 I031H02 1281 143-255 165-178 194-200 233-244 1-126 I031H03 1282 148-260 170-183 199-205 238-249 1-131 I031H06 1283 145-257 167-179 195-201 234-246 1-128 I031H09 1284 145-255 167-179 195-201 234-244 1-128 I031H10 1285 144-256 166-179 195-201 234-245 1-127 I031H11 1286 136-246 158-170 186-192 225-235 1-119 I033A08 1287 144-254 166-179 195-201 234-243 1-128 I033B11 1288 144-254 166-179 195-201 234-243 1-128 I033C01 1289 144-254 166-179 195-201 234-243 1-128 I033C08 1290 142-249 163-173 189-195 228-238 1-126 I033D02 1291 138-245 161-171 187-193 226-234 1-122 I033D03 1292 141-251 163-176 192-198 231-240 1-125 I033D05 1293 141-248 162-172 188-194 227-237 1-125 I033D11 1294 140-247 161-171 187-193 226-236 1-124 I033D12 1295 144-254 166-179 195-201 234-243 1-128 I033E01 1296 139-249 161-173 189-195 228-238 1-123 I033E06 1297 141-251 163-176 192-198 231-240 1-125 I033E11 1298 143-253 165-177 193-199 232-242 1-127 I033E12 1299 142-249 163-173 189-195 228-238 1-126 I033F03 1300 139-246 160-170 186-192 225-235 1-123 I033F08 1301 145-256 167-179 195-201 234-245 1-129 I033F10 1302 144-254 166-179 195-201 234-243 1-128 I033F12 1303 134-241 155-165 181-187 220-230 1-118 I033G01 1304 143-253 165-178 194-200 233-242 1-127 I033G03 1305 142-249 163-173 189-195 228-238 1-126 I033G08 1306 141-248 162-172 188-194 227-237 1-125 I033H04 1307 140-247 161-171 187-193 226-236 1-124 I037A05 1308 139-246 160-170 186-192 225-235 1-123 I037B03 1309 141-251 163-175 191-197 230-240 1-125 I037B04 1310 144-251 167-177 193-199 232-240 1-128 I037C04 1311 142-252 164-177 193-199 232-241 1-126 I037C06 1312 141-249 163-173 189-195 228-238 1-125 I037C08 1313 140-250 162-175 191-197 230-239 1-124 I037D11 1314 136-246 158-171 187-193 226-235 1-120 I037E06 1315 144-251 165-175 191-197 230-240 1-128 I037F04 1316 144-251 165-175 191-197 230-240 1-128 I037G01 1317 141-251 163-176 192-198 231-240 1-125 I037G03 1318 146-256 168-181 197-203 236-245 1-130 I037G10 1319 140-250 162-175 191-197 230-239 1-124 I042A07 1320 144-251 167-177 193-199 232-240 1-128 I042A10 1321 142-249 165-175 191-197 230-238 1-126 I042B03 1322 140-247 161-171 187-193 226-236 1-124 I042B12 1323 141-248 162-172 188-194 227-237 1-125 I042D01 1324 136-246 158-171 187-193 226-235 1-120 I042D03 1325 140-250 162-175 191-197 230-239 1-124 I042D10 1326 142-252 164-177 193-199 232-241 1-126 I042E10 1327 147-257 169-182 198-204 237-246 1-131 I042E11 1328 140-247 161-171 187-193 226-236 1-124 I042F08 1329 142-252 164-177 193-199 232-241 1-126 I042F12 1330 140-247 161-171 187-193 226-236 1-124 I042G08 1331 141-248 162-172 188-194 227-237 1-125 I042G10 1332 141-251 163-176 192-198 231-240 1-125 I042H03 1333 143-253 165-178 194-200 233-242 1-127 I043A03 1334 144-254 166-179 195-201 234-243 1-128 I043B02 1335 142-249 163-173 189-195 228-238 1-126 I043B03 1336 141-251 163-176 192-198 231-240 1-125 I043B06 1337 143-253 165-178 194-200 233-242 1-127 I043B07 1338 141-251 163-176 192-198 231-240 1-125 I043B09 1339 143-253 165-178 194-200 233-242 1-127 I043D11 1340 144-254 166-179 195-201 234-243 1-128 I043E05 1341 143-250 164-174 190-196 229-239 1-127 I043F01 1342 141-251 163-176 192-198 231-240 1-125 I043F04 1343 141-251 163-176 192-198 231-240 1-125 I043F12 1344 143-250 164-174 190-196 229-239 1-127 I043H07 1345 141-251 163-176 192-198 231-240 1-125 I044A11 1346 144-251 165-175 191-197 230-240 1-128 I044B11 1347 139-249 161-173 189-195 228-238 1-123 I044C09 1348 140-250 162-174 190-196 229-239 1-124 I044C10 1349 143-253 165-177 193-199 232-242 1-127 I044D03 1350 144-254 166-179 195-201 234-243 1-128 I044D09 1351 141-251 163-176 192-198 231-240 1-125 I044E07 1352 137-247 159-172 188-194 227-236 1-121 I044E11 1353 143-253 165-178 194-200 233-242 1-127 I044F07 1354 147-257 169-182 198-204 237-246 1-131 I044G02 1355 141-251 163-176 192-198 231-240 1-125 I044G07 1356 149-259 171-184 200-206 239-248 1-133 I044H01 1357 144-251 165-175 191-197 230-240 1-128 I050A01 1358 142-253 164-177 193-199 232-242 1-125 I050B12 1359 142-253 164-177 193-199 232-242 1-125 I050006 1360 142-248 165-175 191-197 230-237 1-124 I050008 1361 142-253 164-177 193-199 232-242 1-125 I050E01 1362 141-252 163-176 192-198 231-241 1-124 I050E10 1363 138-248 160-172 188-194 227-237 1-121 I050H08 1364 142-253 164-177 193-199 232-242 1-125 I051A04 1365 148-258 170-183 199-205 238-247 1-131 I051A08 1366 142-252 164-176 192-198 231-241 1-125 I051A12 1367 143-250 164-174 190-196 229-239 1-127 I051B08 1368 143-253 165-177 193-199 232-242 1-126 I051C06 1369 136-246 158-170 186-192 225-235 1-119 I051G12 1370 143-250 164-174 190-196 229-239 1-127 I055A05 1371 134-244 156-169 185-191 224-233 1-117 I055A11 1372 134-244 156-169 185-191 224-233 1-117 I061A03 1373 141-251 163-176 192-198 231-240 1-124 I061A04 1374 143-251 165-175 191-197 230-240 1-125 I061A08 1375 142-253 164-176 192-198 233-242 1-124 I061A09 1376 142-252 164-176 192-198 231-241 1-124 I061A10 1377 141-249 163-173 189-195 228-238 1-124 I061B07 1378 141-252 163-176 192-198 231-241 1-124 I061B09 1379 143-253 165-178 194-200 233-242 1-127 I061B12 1380 141-251 163-176 192-198 231-240 1-125 I061C12 1381 138-248 160-173 189-195 228-237 1-122 I061D01 1382 137-247 159-172 188-194 227-236 1-121 I061D03 1383 141-251 163-176 192-198 231-240 1-125 I061D04 1384 140-247 161-171 187-193 226-236 1-124 I061D07 1385 141-248 164-174 190-196 229-237 1-125 I061D09 1386 141-248 162-172 188-194 227-237 1-125 I061D10 1387 141-248 162-172 188-194 227-237 1-125 I061E01 1388 141-248 162-172 188-194 227-237 1-125 I061E05 1389 142-251 163-175 191-197 230-240 1-126 I061E09 1390 141-251 163-176 192-198 231-240 1-125 I061E12 1391 133-240 154-164 180-186 219-229 1-117 I061F01 1392 146-256 168-181 197-203 236-245 1-130 I061F09 1393 139-246 160-170 186-192 225-235 1-123 I061F10 1394 145-252 166-176 192-198 231-241 1-129 I061F11 1395 141-251 163-176 192-198 231-240 1-125 I061G01 1396 141-251 163-176 192-198 231-240 1-125 I061G03 1397 141-251 163-176 192-198 231-240 1-125 I061G09 1398 144-254 166-179 195-201 234-243 1-128 I061G10 1399 143-253 165-178 194-200 233-242 1-127 I061G11 1400 137-247 159-171 187-193 226-236 1-121 I061H05 1401 142-252 164-177 193-199 232-241 1-126 I064A05 1402 142-249 163-173 189-195 228-238 1-126 I064A11 1403 138-248 160-173 189-195 228-237 1-122 I064B01 1404 138-248 160-173 189-195 228-237 1-122 I064B02 1405 144-254 166-179 195-201 234-243 1-128 I064B12 1406 133-243 155-168 184-190 223-232 1-117 I064C06 1407 145-255 167-180 196-202 235-244 1-129 I064D01 1408 141-251 163-176 192-198 231-240 1-125 I064D02 1409 146-256 168-181 197-203 236-245 1-130 I064E01 1410 143-250 166-176 192-198 231-239 1-127 I064E02 1411 140-251 162-174 190-196 229-240 1-124 I064E03 1412 144-254 166-179 195-201 234-243 1-128 I064E07 1413 140-250 162-175 191-197 230-239 1-124 I064E08 1414 140-250 162-174 190-196 229-239 1-124 I064F05 1415 142-252 164-177 193-199 232-241 1-126 I064F08 1416 145-252 166-176 192-198 231-241 1-129 I064G06 1417 141-248 162-172 188-194 227-237 1-125 I065A12 1418 143-253 165-178 194-200 233-242 1-127 I065C04 1419 139-249 161-173 189-195 228-238 1-123 I065C09 1420 140-250 162-174 190-196 229-239 1-124 I065E02 1421 141-248 164-174 190-196 229-237 1-125 I065E04 1422 135-245 157-169 185-191 224-234 1-119 I065F03 1423 137-247 159-172 188-194 227-236 1-121 I065G06 1424 135-242 156-166 182-188 221-231 1-119 I065G07 1425 142-249 163-173 189-195 228-238 1-126 I065G08 1426 139-246 160-170 186-192 225-235 1-123 I065H06 1427 144-254 166-179 195-201 234-243 1-128 I066A03 1428 144-254 166-179 195-201 234-243 1-128 I066A08 1429 137-247 159-171 187-193 226-236 1-121 I066A09 1430 135-245 157-169 185-191 224-234 1-119 I066A10 1431 142-252 164-177 193-199 232-241 1-126 I066A11 1432 143-253 165-178 194-200 233-242 1-127 I066B02 1433 135-242 156-166 182-188 221-231 1-119 I066B08 1434 137-247 159-172 188-194 227-236 1-121 I066B10 1435 142-252 164-177 193-199 232-241 1-126 I066C02 1436 135-245 157-169 185-191 224-234 1-119 I066C11 1437 141-251 163-176 192-198 231-240 1-125 I066C12 1438 135-242 156-166 182-188 221-231 1-119 I066D06 1439 140-250 162-175 191-197 230-239 1-124 I066D08 1440 138-248 160-173 189-195 228-237 1-122 I066D11 1441 144-254 166-179 195-201 234-243 1-128 I066D12 1442 139-249 161-174 190-196 229-238 1-123 I066E06 1443 137-247 159-171 187-193 226-236 1-121 I066E12 1444 135-242 156-166 182-188 221-231 1-119 I066G05 1445 142-249 163-173 189-195 228-238 1-126 I066G08 1446 141-248 164-174 190-196 229-237 1-125 I066G10 1447 144-254 166-178 194-200 233-243 1-128 I066G12 1448 143-254 165-177 193-199 232-243 1-127 I066H04 1449 143-253 165-178 194-200 233-242 1-127 I067A07 1450 144-254 166-179 195-201 234-243 1-128 I067A11 1451 135-245 157-170 186-192 225-234 1-119 I067B08 1452 149-259 171-184 200-206 239-248 1-133 I067C08 1453 148-258 170-183 199-205 238-247 1-132 I067C09 1454 143-253 165-178 194-200 233-242 1-127 I067D07 1455 137-247 159-171 187-193 226-236 1-121 I067E01 1456 142-248 164-174 190-196 229-238 1-124 I067E06 1457 135-245 157-169 185-191 224-234 1-119 I067E07 1458 150-260 172-184 200-206 239-249 1-134 I067E11 1459 141-251 163-176 192-198 231-240 1-125 I067G03 1460 140-250 162-175 191-197 230-239 1-124 I067G05 1461 140-250 162-174 190-196 229-239 1-124 I067G12 1462 141-252 163-176 192-198 231-241 1-125 I067H05 1463 146-256 168-180 196-202 235-245 1-130 I067H06 1464 135-245 157-169 185-191 224-234 1-119 I068C09 1465 138-248 160-172 188-194 227-237 1-121 I068G03 1466 144-254 166-178 194-200 233-243 1-127 I068G04 1467 143-252 165-178 194-200 233-241 1-126 I068G07 1468 142-251 164-174 190-196 229-240 1-124 I068G08 1469 144-254 166-178 194-200 233-243 1-127 I070F07 1470 140-247 161-171 187-193 226-236 1-124 I070G05 1471 140-250 162-175 191-197 230-239 1-124 I070H02 1472 141-248 162-172 188-194 227-237 1-125 I071A01 1473 141-251 163-176 192-198 231-240 1-125 I071A03 1474 143-250 164-174 190-196 229-239 1-127 I071B08 1475 144-252 166-176 192-198 231-241 1-126 I071E01 1476 138-248 160-173 189-195 228-237 1-122 I071F11 1477 135-245 157-169 185-191 224-234 1-119 I071G11 1478 141-251 163-176 192-198 231-240 1-125 I071H08 1479 141-251 163-176 192-198 231-240 1-125 I074A02 1480 142-250 164-174 190-196 229-239 1-125 I074A08 1481 148-259 170-182 198-204 237-248 1-131 I074D10 1482 146-253 168-178 194-200 233-242 1-128 I074E01 1483 146-255 168-178 194-200 233-244 1-128 I074E02 1484 142-250 164-174 190-196 229-239 1-124 I074E08 1485 144-259 166-179 195-205 240-248 1-127 I074F12 1486 142-250 164-174 190-196 229-239 1-124 I074H06 1487 140-251 162-175 191-197 230-240 1-123 I074H07 1488 145-253 167-177 193-199 232-242 1-127 I074H08 1489 143-254 165-178 194-200 233-243 1-126 I075A07 1490 145-253 167-177 193-199 232-242 1-127 I075B01 1491 134-244 156-168 184-190 223-233 1-117 I075B04 1492 134-247 156-169 185-191 224-236 1-117 I075B06 1493 141-252 163-175 191-197 230-241 1-124 I075B08 1494 144-257 166-179 195-201 234-246 1-127 I075B09 1495 142-252 164-176 192-198 231-241 1-125 I075B12 1496 141-251 163-176 192-198 231-240 1-124 I075C01 1497 148-259 170-183 199-205 238-248 1-131 I075C05 1498 134-244 156-168 184-190 223-233 1-117 I075D05 1499 145-253 168-179 195-201 234-242 1-127 I075D07 1500 142-252 164-176 192-198 231-241 1-125 I075D08 1501 141-251 163-175 191-197 230-240 1-124 I075E01 1502 145-253 167-177 193-199 232-242 1-127 I075E03 1503 150-261 172-184 200-206 239-250 1-132 I075E04 1504 144-255 166-179 195-201 234-244 1-127 I075E05 1505 141-252 163-176 192-198 231-241 1-124 I075E10 1506 141-252 163-176 192-198 231-241 1-124 I075E11 1507 134-244 156-168 184-190 223-233 1-117 I075E12 1508 143-254 165-178 194-200 233-243 1-126 I075F02 1509 146-253 168-178 194-200 233-242 1-128 I075F04 1510 142-251 164-176 192-198 231-240 1-125 I075F06 1511 146-254 168-178 194-200 233-243 1-128 I075F07 1512 141-251 163-175 191-197 230-240 1-124 I075F08 1513 134-244 156-168 184-190 223-233 1-117 I075F09 1514 147-257 169-181 197-203 236-246 1-129 I075F10 1515 135-243 157-167 183-189 222-232 1-117 I075F11 1516 134-245 156-169 185-191 224-234 1-117 I075G05 1517 141-252 163-175 191-197 230-241 1-124 I075G07 1518 141-252 163-175 191-197 230-241 1-124 I075G08 1519 141-252 163-176 192-198 231-241 1-124 I075G11 1520 142-253 164-177 193-199 232-242 1-125 I075G12 1521 134-245 156-169 185-191 224-234 1-117 I075H02 1522 144-254 166-178 194-200 233-243 1-127 I075H03 1523 134-245 156-169 185-191 224-234 1-117 I075H06 1524 134-244 156-168 184-190 223-233 1-117 I075H08 1525 144-254 166-179 195-201 234-243 1-127 I076A01 1526 144-253 166-176 192-198 231-242 1-126 I076A03 1527 137-247 159-171 187-193 226-236 1-119 I076A06 1528 134-245 156-168 184-190 223-234 1-117 I076A07 1529 140-250 162-174 190-196 229-239 1-123 I076A08 1530 144-253 166-176 192-198 231-242 1-126 I076B01 1531 145-257 167-179 195-201 236-246 1-127 I076B03 1532 134-245 156-169 185-191 224-234 1-117 I076B07 1533 135-243 157-167 183-189 222-232 1-117 I076B08 1534 143-252 166-177 193-199 232-241 1-125 I076C04 1535 142-250 164-174 190-196 229-239 1-124 I076C10 1536 141-251 163-175 191-197 230-240 1-124 I076D01 1537 142-252 164-176 192-198 231-241 1-125 I076D08 1538 141-251 163-175 191-197 230-240 1-124 I076D11 1539 144-255 166-179 195-201 234-244 1-127 I076D12 1540 142-250 164-174 190-196 229-239 1-124 I076E04 1541 145-252 167-177 193-199 232-241 1-127 I076E07 1542 141-251 163-175 191-197 230-240 1-124 I076E09 1543 142-253 164-177 193-199 232-242 1-125 I076E11 1544 144-254 166-179 195-201 234-243 1-127 I076F01 1545 144-253 166-178 194-199 232-242 1-127 I076F03 1546 141-251 163-175 191-197 230-240 1-124 I076F04 1547 135-245 157-169 185-191 224-234 1-117 I076F08 1548 142-250 164-174 190-196 229-239 1-124 I076F10 1549 141-252 163-175 191-197 230-241 1-124 I076G09 1550 134-245 156-168 184-190 223-234 1-117 I076G10 1551 141-251 163-175 191-197 230-240 1-124 I076G11 1552 144-259 166-179 195-205 240-248 1-127 I076G12 1553 147-257 169-181 197-203 236-246 1-130 I076H02 1554 141-251 163-175 191-197 230-240 1-124 I076H04 1555 143-251 165-175 191-197 230-240 1-125 I076H05 1556 141-251 163-175 191-197 230-240 1-124 I076H06 1557 141-252 163-176 192-198 231-241 1-124 I076H09 1558 144-256 166-179 195-201 234-245 1-127 I076H10 1559 144-256 166-179 195-201 234-245 1-127 I077D06 1560 140-250 162-175 191-197 230-239 1-124 I078B04 1561 140-250 162-175 191-197 230-239 1-124 I078E10 1562 141-251 163-176 192-198 231-240 1-125 I002A01-K 1563 142-250 164-174 190-196 229-239 1-125 I002A01-R 1564 142-250 164-174 190-196 229-239 1-125 I026C04-K 1565 142-250 164-176 192-198 231-239 1-125 I026C04-R 1566 142-250 164-176 192-198 231-239 1-125 I067B10 1567 149-259 171-183 199-205 238-248 1-133 I068C06 1568 134-244 156-169 185-191 224-233 1-117 I075F12 1569 134-244 156-168 184-190 223-233 1-117 I003C06 1570 141-249 163-173 189-195 228-238 1-124 I025B06 1571 141-249 163-175 191-197 230-238 1-124 I025B09 1572 141-249 163-175 191-197 230-238 1-124 I026C04 1573 141-249 163-175 191-197 230-238 1-124 I027B12 1574 142-250 164-174 190-196 229-239 1-125 I030A10 1575 141-252 163-176 192-198 231-241 1-124 I064C04 1576 147-257 169-182 198-204 237-246 1-131 I064C07 1577 134-241 157-167 183-189 222-230 1-118 I065D04 1578 144-254 166-179 195-201 234-243 1-128 I065D08 1579 147-257 169-182 198-204 237-246 1-131 I065F08 1580 135-242 158-168 184-190 223-231 1-119 I067F05 1581 140-250 162-175 191-197 230-239 1-124 I068B04 1582 134-244 156-168 184-190 223-233 1-117 I068B08 1583 141-252 163-175 191-197 231-241 1-124 I068C08 1584 143-254 165-178 194-200 233-243 1-126 I068F03 1585 140-251 162-175 191-197 230-240 1-123 I069B07 1586 141-251 163-176 192-198 231-240 1-125 I071B03 1587 141-251 163-176 192-198 231-240 1-125 I072B09 1588 141-248 162-172 188-194 227-237 1-125 I073F04 1589 136-246 158-171 187-193 226-235 1-120 I074B12 1590 142-252 164-176 192-198 231-241 1-124 I075A02 1591 141-251 163-175 191-197 230-240 1-124 I075G01 1592 142-251 164-174 190-196 229-240 1-124 I078D02 1593 140-250 162-175 191-197 230-239 1-124 I078D08 1594 144-251 165-175 191-197 230-240 1-128 I078H08 1595 140-250 162-175 191-197 230-239 1-124 I064A03 1596 150-257 171-181 197-203 236-246 1-134 I064B03 1597 145-255 167-179 195-201 234-244 1-129 I064B05 1598 140-250 162-174 190-196 229-239 1-124 I064B11 1599 138-248 160-173 189-195 228-237 1-122 I064C02 1600 146-256 168-180 196-202 235-245 1-130 I064C03 1601 140-250 162-175 191-197 230-239 1-124 I064C11 1602 143-253 165-178 194-200 233-242 1-127 I064C12 1603 148-255 171-181 197-203 236-244 1-132 I064D03 1604 146-256 168-181 197-203 236-245 1-130 I064D04 1605 141-251 163-176 192-198 231-240 1-125 I064D06 1606 134-244 156-169 185-191 224-233 1-118 I064E05 1607 146-256 168-180 196-202 235-245 1-130 I064E06 1608 145-255 167-180 196-202 235-244 1-129 I064F07 1609 141-248 162-172 188-194 227-237 1-125 I064F09 1610 147-257 169-181 197-203 236-246 1-131 I064F10 1611 143-253 165-177 193-199 232-242 1-127 I064F11 1612 142-252 164-177 193-199 232-241 1-126 I064G01 1613 140-250 162-175 191-197 230-239 1-124 I064G04 1614 133-243 155-167 183-189 222-232 1-117 I064G08 1615 138-245 159-169 185-191 224-234 1-122 I064G10 1616 140-250 162-175 191-197 230-239 1-124 I064G11 1617 138-248 160-173 189-195 228-237 1-122 I064G12 1618 139-249 161-173 189-195 228-238 1-123 I064H03 1619 143-253 165-178 194-200 233-242 1-127 I064H04 1620 142-249 163-173 189-195 228-238 1-126 I064H06 1621 149-256 170-180 196-202 235-245 1-133 I065A02 1622 141-248 162-172 188-194 227-237 1-125 I065A04 1623 141-248 162-172 188-194 227-237 1-125 I065A06 1624 141-248 162-172 188-194 227-237 1-125 I065A07 1625 144-254 166-179 195-201 234-243 1-128 I065B01 1626 145-255 167-180 196-202 235-244 1-129 I065B05 1627 142-252 164-177 193-199 232-241 1-126 I065B09 1628 146-253 167-177 193-199 232-242 1-130 I065B12 1629 139-249 161-174 190-196 229-238 1-123 I065C02 1630 136-246 158-170 186-192 225-235 1-120 I065C06 1631 141-253 163-175 191-197 230-242 1-125 I065C08 1632 141-250 163-176 192-198 231-239 1-125 I065C10 1633 137-247 159-172 188-194 227-236 1-121 I065D01 1634 142-252 164-177 193-199 232-241 1-126 I065D03 1635 142-249 165-175 191-197 230-238 1-126 I065D05 1636 143-253 165-178 194-200 233-242 1-127 I065D06 1637 142-252 164-177 193-199 232-241 1-126 I065E01 1638 139-246 160-170 186-192 225-235 1-123 I065E05 1639 137-244 158-168 184-190 223-233 1-121 I065E06 1640 146-256 168-181 197-203 236-245 1-130 I065E08 1641 142-249 163-173 189-195 228-238 1-126 I065E09 1642 145-255 167-179 195-201 234-244 1-129 I065E12 1643 141-248 162-172 188-194 227-237 1-125 I065F04 1644 140-250 162-175 191-197 230-239 1-124 I065F05 1645 140-250 162-175 191-197 230-239 1-124 I065F07 1646 145-252 166-176 192-198 231-241 1-129 I065F09 1647 143-250 164-174 190-196 229-239 1-127 I065F12 1648 141-248 162-172 188-194 227-237 1-125 I065G01 1649 141-251 163-176 192-198 231-240 1-125 I065G09 1650 143-253 165-178 194-200 233-242 1-127 I065G10 1651 140-247 161-171 187-193 226-236 1-124 I065H05 1652 140-247 161-171 187-193 226-236 1-124 I065H07 1653 138-248 160-173 189-195 228-237 1-122 I066A05 1654 137-247 159-172 188-194 227-236 1-121 I066A06 1655 139-246 160-170 186-192 225-235 1-123 I066A12 1656 142-252 164-177 193-199 232-241 1-126 I066B05 1657 141-248 162-172 188-194 227-237 1-125 I066B11 1658 142-252 164-177 193-199 232-241 1-126 I066C06 1659 144-254 166-178 194-200 233-243 1-128 I066C10 1660 141-248 162-172 188-194 227-237 1-125 I066D02 1661 137-247 159-172 188-194 227-236 1-121 I066D07 1662 141-248 162-172 188-194 227-237 1-125 I066E01 1663 137-247 159-172 188-194 227-236 1-121 I066E03 1664 149-259 171-184 200-206 239-248 1-133 I066E04 1665 141-248 162-172 188-194 227-237 1-125 I066E05 1666 142-252 164-177 193-199 232-241 1-126 I066E07 1667 141-248 162-172 188-194 227-237 1-125 I066E09 1668 141-248 162-172 188-194 227-237 1-125 I066F01 1669 141-251 163-176 192-198 231-240 1-125 I066F03 1670 141-248 162-172 188-194 227-237 1-125 I066F04 1671 141-251 163-175 191-197 230-240 1-125 I066F07 1672 143-253 165-178 194-200 233-242 1-127 I066F08 1673 144-254 166-179 195-201 234-243 1-128 I066F11 1674 142-252 164-177 193-199 232-241 1-126 I066F12 1675 141-248 162-172 188-194 227-237 1-125 I066G06 1676 143-250 164-174 190-196 229-239 1-127 I066G07 1677 133-243 155-168 184-190 223-232 1-117 I066H02 1678 135-242 156-166 182-188 221-231 1-119 I067A02 1679 141-248 162-172 188-194 227-237 1-125 I067A03 1680 137-247 159-172 188-194 227-236 1-121 I067A06 1681 141-248 162-172 188-194 227-237 1-125 I067A08 1682 137-247 159-171 187-193 226-236 1-121 I067A10 1683 140-250 162-175 191-197 230-239 1-124 I067B03 1684 142-253 164-177 193-199 232-242 1-126 I067B04 1685 137-247 159-172 188-194 227-236 1-121 I067C03 1686 134-244 156-169 185-191 224-233 1-117 I067C05 1687 137-247 159-172 188-194 227-236 1-121 I067C07 1688 141-251 163-176 192-198 231-240 1-125 I067C10 1689 137-247 159-172 188-194 227-236 1-121 I067C12 1690 142-252 164-177 193-199 232-241 1-126 I067D01 1691 136-246 158-171 187-193 226-235 1-120 I067D03 1692 137-244 158-168 184-190 223-233 1-121 I067D05 1693 146-256 168-180 196-202 235-245 1-130 I067D06 1694 137-244 158-168 184-190 223-233 1-121 I067D09 1695 142-252 164-177 193-199 232-241 1-126 I067D12 1696 137-247 159-172 188-194 227-236 1-121 I067E02 1697 137-247 159-172 188-194 227-236 1-121 I067E04 1698 142-252 164-176 192-198 231-241 1-126 I067E05 1699 144-254 166-179 195-201 234-243 1-128 I067F01 1700 141-248 164-174 190-196 229-237 1-125 I067F03 1701 144-254 166-179 195-201 234-243 1-128 I067F04 1702 139-246 160-170 186-192 225-235 1-123 I067F08 1703 140-247 161-171 187-193 226-236 1-124 I067F10 1704 137-247 159-172 188-194 227-236 1-121 I067F11 1705 140-248 161-171 187-193 226-237 1-124 I067G01 1706 141-251 163-176 192-198 231-240 1-125 I067G09 1707 137-247 159-171 187-193 226-236 1-121 I067H07 1708 144-251 165-175 191-197 230-240 1-128 I068A07 1709 143-254 165-178 194-200 233-243 1-126 I068E05 1710 148-257 170-183 199-205 238-246 1-131 I068E08 1711 135-247 157-169 185-193 226-236 1-117 I068E11 1712 141-251 163-176 192-198 231-240 1-124 I068F04 1713 142-252 164-176 192-198 231-241 1-125 I068G05 1714 137-245 159-169 185-191 224-234 1-119 I068G06 1715 140-250 162-174 190-196 229-239 1-123 I068G11 1716 147-258 169-182 198-204 237-247 1-130 I069A09 1717 141-248 164-174 190-196 229-237 1-125 I069A10 1718 141-248 162-172 188-194 227-237 1-125 I069B06 1719 141-248 164-174 190-196 229-237 1-125 I069B09 1720 139-249 161-174 190-196 229-238 1-123 I069B12 1721 141-248 162-172 188-194 227-237 1-125 I069C06 1722 143-250 164-174 190-196 229-239 1-127 I069C09 1723 143-250 164-174 190-196 229-239 1-127 I069D03 1724 142-249 163-173 189-195 228-238 1-126 I069E09 1725 142-249 163-173 189-195 228-238 1-126 I069E11 1726 140-247 161-171 187-193 226-236 1-124 I069F05 1727 141-248 162-172 188-194 227-237 1-125 I069F07 1728 141-248 162-172 188-194 227-237 1-125 I069F12 1729 140-247 161-171 187-193 226-236 1-124 I069G06 1730 142-249 163-173 189-195 228-238 1-126 I069G08 1731 145-252 166-176 192-198 231-241 1-129 I069G11 1732 141-248 162-172 188-194 227-237 1-125 I070A03 1733 141-248 164-174 190-196 229-237 1-125 I070A09 1734 141-248 162-172 188-194 227-237 1-125 I070B01 1735 144-254 166-179 195-201 234-243 1-128 I070B05 1736 141-251 163-176 192-198 231-240 1-125 I070D03 1737 141-248 164-174 190-196 229-237 1-125 I070D04 1738 141-251 163-176 192-198 231-240 1-125 I070E01 1739 144-254 166-179 195-201 234-243 1-128 I070F01 1740 144-251 165-175 191-197 230-240 1-128 I070G10 1741 141-248 162-172 188-194 227-237 1-125 I071A06 1742 135-242 156-166 182-188 221-231 1-119 I071B02 1743 135-245 157-170 186-192 225-234 1-119 I071D02 1744 137-247 159-172 188-194 227-236 1-121 I071D08 1745 146-256 168-181 197-203 236-245 1-130 I071F01 1746 137-247 159-172 188-194 227-236 1-121 I071G09 1747 141-251 163-176 192-198 231-240 1-125 I072A01 1748 139-249 161-174 190-196 229-238 1-123 I072A09 1749 141-251 163-176 192-198 231-240 1-125 I072B02 1750 135-245 157-170 186-192 225-234 1-119 I072B10 1751 137-247 159-172 188-194 227-236 1-121 I072B11 1752 141-251 163-176 192-198 231-240 1-125 I072B12 1753 140-249 162-173 189-195 228-238 1-124 I072C05 1754 135-245 157-169 185-191 224-234 1-119 I072C10 1755 141-248 162-172 188-194 227-237 1-125 I072D01 1756 141-251 163-176 192-198 231-240 1-125 I072D05 1757 135-245 157-169 185-191 224-234 1-119 I072E01 1758 141-251 163-176 192-198 231-240 1-125 I072E04 1759 144-254 166-179 195-201 234-243 1-128 I072E05 1760 141-251 163-176 192-198 231-240 1-125 I072E06 1761 135-242 156-166 182-188 221-231 1-119 I072F03 1762 135-242 156-166 182-188 221-231 1-119 I072F07 1763 141-251 163-176 192-198 231-240 1-125 I072F11 1764 140-247 161-171 187-193 226-236 1-124 I072G03 1765 141-248 162-172 188-194 227-237 1-125 I072G04 1766 137-247 159-171 187-193 226-236 1-121 I072G05 1767 137-247 159-171 187-193 226-236 1-121 I072G09 1768 141-251 163-176 192-198 231-240 1-125 I072H03 1769 141-251 163-176 192-198 231-240 1-125 I072H07 1770 137-247 159-172 188-194 227-236 1-121 I073A02 1771 141-248 164-174 190-196 229-237 1-125 I073A03 1772 142-252 164-177 193-199 232-241 1-126 I073A04 1773 148-258 170-183 199-205 238-247 1-132 I073A05 1774 141-251 163-176 192-198 231-240 1-125 I073A06 1775 141-251 163-176 192-198 231-240 1-125 I073A09 1776 141-251 163-176 192-198 231-240 1-125 I073A10 1777 146-253 167-177 193-199 232-242 1-130 I073A11 1778 141-248 164-174 190-196 229-237 1-125 I073B02 1779 144-254 166-179 195-201 234-243 1-128 I073B05 1780 144-254 166-179 195-201 234-243 1-128 I073B06 1781 139-246 160-170 186-192 225-235 1-123 I073B07 1782 138-248 160-173 189-195 228-237 1-122 I073B08 1783 141-251 163-176 192-198 231-240 1-125 I073B11 1784 141-251 163-176 192-198 231-240 1-125 I073C01 1785 141-248 162-172 188-194 227-237 1-125 I073C02 1786 148-255 169-179 195-201 234-244 1-132 I073C04 1787 142-252 164-177 193-199 232-241 1-125 I073C07 1788 134-241 155-165 181-187 220-230 1-118 I073C08 1789 142-252 164-177 193-199 232-241 1-126 I073C09 1790 141-248 162-172 188-194 227-237 1-125 I073C11 1791 146-256 168-181 197-203 236-245 1-130 I073C12 1792 141-251 163-176 192-198 231-240 1-125 I073D01 1793 141-251 163-176 192-198 231-240 1-125 I073D03 1794 135-245 157-169 185-191 224-234 1-119 I073D06 1795 141-251 163-176 192-198 231-240 1-125 I073D08 1796 144-254 166-179 195-201 234-243 1-128 I073D10 1797 140-250 162-175 191-197 230-239 1-124 I073D11 1798 141-251 163-176 192-198 231-240 1-125 I073E01 1799 148-258 170-183 199-205 238-247 1-132 I073E02 1800 141-251 163-176 192-198 231-240 1-125 I073E03 1801 141-251 163-176 192-198 231-240 1-125 I073E05 1802 141-251 163-176 192-198 231-240 1-125 I073E06 1803 141-251 163-176 192-198 231-240 1-125 I073E08 1804 140-250 162-175 191-197 230-239 1-124 I073F01 1805 141-251 163-175 191-197 230-240 1-125 I073F02 1806 141-251 163-175 191-197 230-240 1-125 I073F03 1807 141-251 163-176 192-198 231-240 1-125 I073F05 1808 141-251 163-176 192-198 231-240 1-125 I073F07 1809 141-251 163-175 191-197 230-240 1-125 I073F09 1810 141-251 163-176 192-198 231-240 1-125 I073F11 1811 141-251 163-176 192-198 231-240 1-125 I073F12 1812 141-251 163-175 191-197 230-240 1-125 I073G03 1813 143-253 165-178 194-200 233-242 1-127 I073G04 1814 143-253 165-178 194-200 233-242 1-127 I073G05 1815 135-245 157-169 185-191 224-234 1-119 I073G06 1816 141-248 162-172 188-194 227-237 1-125 I073G07 1817 142-249 163-173 189-195 228-238 1-126 I073G08 1818 139-246 160-170 186-192 225-235 1-123 I073G09 1819 145-255 167-180 196-202 235-244 1-129 I073G10 1820 135-245 157-170 186-192 225-234 1-119 I073G12 1821 142-252 164-177 193-199 232-241 1-126 I073H01 1822 141-251 163-176 192-198 231-240 1-125 I073H03 1823 141-248 162-172 188-194 227-237 1-125 I073H05 1824 141-251 163-176 192-198 231-240 1-125 I073H06 1825 141-251 163-176 192-198 231-240 1-125 I073H07 1826 138-245 159-169 185-191 224-234 1-122 I073H08 1827 141-251 163-176 192-198 231-240 1-125 I074A05 1828 144-255 166-179 195-201 234-244 1-127 I074A06 1829 145-253 167-177 193-199 232-242 1-127 I074B03 1830 134-242 156-166 182-188 221-231 1-117 I074B11 1831 140-251 162-175 191-197 230-240 1-123 I074C07 1832 141-251 163-175 191-197 230-240 1-124 I074D03 1833 143-251 165-175 191-197 230-240 1-125 I074D04 1834 134-246 156-169 185-191 224-235 1-117 I074D05 1835 145-253 167-177 193-199 232-242 1-127 I074D07 1836 151-262 173-186 202-208 241-251 1-134 I074D08 1837 141-251 163-175 191-197 230-240 1-124 I074D11 1838 139-249 161-174 190-196 229-238 1-122 I074E05 1839 134-245 156-169 185-191 224-234 1-117 I074E07 1840 141-251 163-175 191-197 230-240 1-124 I074E09 1841 147-258 169-182 198-204 237-247 1-130 I074E11 1842 138-244 160-170 186-192 225-233 1-121 I074H05 1843 144-254 166-178 194-200 233-243 1-126 I075A03 1844 135-242 158-168 184-190 223-231 1-117 I075A10 1845 135-244 157-169 185-191 224-233 1-117 I075B07 1846 144-254 166-178 194-200 233-243 1-127 I075D11 1847 134-246 156-169 185-191 224-235 1-117 I075D12 1848 145-253 167-177 193-199 232-242 1-127 I075G02 1849 144-255 166-179 195-201 234-244 1-127 I075G09 1850 143-253 165-177 193-199 232-242 1-126 I075G10 1851 140-250 162-174 190-196 229-239 1-122 I075H05 1852 142-252 164-176 192-198 231-241 1-125 I075H07 1853 145-253 167-177 193-199 232-242 1-127 I076A11 1854 142-254 164-177 193-199 232-243 1-125 I076A12 1855 144-256 166-178 194-200 233-245 1-127 I076B06 1856 142-249 164-174 190-196 229-238 1-124 I076B10 1857 142-254 164-177 193-199 232-243 1-125 I076B12 1858 145-253 167-177 193-199 232-242 1-127 I076C06 1859 143-253 165-177 193-199 232-242 1-126 I076C11 1860 134-245 156-168 184-190 223-234 1-117 I076D06 1861 141-252 163-176 192-198 231-241 1-124 I076E05 1862 144-255 166-179 195-201 234-244 1-127 I076E08 1863 135-243 157-167 183-189 222-232 1-117 I076F06 1864 134-245 156-169 185-191 224-234 1-117 I076G01 1865 144-254 166-178 194-200 233-243 1-127 I076H01 1866 146-254 168-178 194-200 233-243 1-128 I076H03 1867 142-250 164-174 190-196 229-239 1-124 I077B05 1868 147-257 169-182 198-204 237-246 1-131 I077C10 1869 141-251 163-176 192-198 231-240 1-125 I077D01 1870 141-251 163-176 192-198 231-240 1-125 I077D04 1871 141-248 162-172 188-194 227-237 1-125 I077D11 1872 141-251 163-176 192-198 231-240 1-125 I077D12 1873 140-247 161-171 187-193 226-236 1-124 I077E01 1874 142-252 164-177 193-199 232-241 1-126 I077E03 1875 142-252 164-177 193-199 232-241 1-126 I077E08 1876 141-248 164-174 190-196 229-237 1-125 I077F05 1877 141-248 162-172 188-194 227-237 1-125 I077G06 1878 141-251 163-176 192-198 231-240 1-125 I077H02 1879 141-248 164-174 190-196 229-237 1-125 I078B05 1880 143-253 165-178 194-200 233-242 1-127 I079E02 1881 137-244 160-170 186-192 225-233 1-121 I079F11 1882 132-239 155-165 181-187 220-228 1-116 I082G02 1883 136-243 159-169 185-191 224-232 1-120 I082H08 1884 132-242 154-167 183-189 222-231 1-115 I099D03 1885 137-247 159-172 188-194 227-236 1-120 I079B05 1886 130-240 152-165 181-187 220-229 1-114 I079B12 1887 134-241 157-167 183-189 222-230 1-118 I079C01 1888 131-241 153-166 182-188 221-230 1-115 I079F06 1889 134-241 157-167 183-189 222-230 1-118 I079F08 1890 138-248 160-172 188-194 227-237 1-122 I080A03 1891 139-249 161-173 189-195 228-238 1-122 I080A08 1892 136-247 158-171 187-193 226-236 1-119 I080B01 1893 144-254 166-178 194-200 233-243 1-126 I080D03 1894 139-249 161-173 189-195 228-238 1-122 I080E05 1895 142-253 164-177 193-199 232-242 1-125 I080G07 1896 138-245 161-172 188-194 227-234 1-120 I080G09 1897 137-249 159-172 188-194 227-238 1-120 I082A05 1898 131-240 153-165 181-187 220-229 1-115 I082B08 1899 137-247 159-171 187-193 226-236 1-121 I082C03 1900 138-245 161-171 187-193 226-234 1-122 I082D07 1901 134-241 157-167 183-189 222-230 1-118 I082G01 1902 138-245 161-171 187-193 226-234 1-122 I083B12 1903 139-247 161-171 187-193 226-236 1-121 I083G03 1904 139-249 161-173 189-195 228-238 1-122 I084A01 1905 130-240 152-164 180-186 219-229 1-114 I084B02 1906 130-237 153-163 179-185 218-226 1-114 I084C04 1907 131-238 152-162 178-184 217-227 1-115 I084C11 1908 134-244 156-169 185-191 224-233 1-118 I079A01 1909 134-243 156-168 184-190 223-232 1-118 I079A03 1910 134-244 156-169 185-191 224-233 1-118 I079A04 1911 134-241 155-165 181-187 220-230 1-118 I079A06 1912 133-240 154-164 180-186 219-229 1-117 I079A07 1913 136-246 158-170 186-192 225-235 1-120 I079A10 1914 148-255 169-179 195-201 234-244 1-132 I079A11 1915 135-242 158-168 184-190 223-231 1-119 I079B02 1916 134-243 156-168 184-190 223-232 1-118 I079B03 1917 136-246 158-170 186-192 225-235 1-120 I079B04 1918 130-240 152-165 181-187 220-229 1-114 I079B07 1919 138-245 159-169 185-191 224-234 1-122 I079B09 1920 139-246 162-172 188-194 227-235 1-123 I079C02 1921 144-251 167-177 193-199 232-240 1-128 I079C04 1922 132-239 155-165 181-187 220-228 1-116 I079C05 1923 140-247 163-173 189-195 228-236 1-124 I079C07 1924 137-244 158-168 184-190 223-233 1-121 I079D01 1925 144-254 166-179 195-201 234-243 1-128 I079D02 1926 135-245 157-169 185-191 224-234 1-119 I079D04 1927 133-243 155-167 183-189 222-232 1-117 I079D06 1928 137-247 159-171 187-193 226-236 1-121 I079D07 1929 136-243 157-167 183-189 222-232 1-120 I079D08 1930 130-240 152-165 181-187 220-229 1-114 I079D09 1931 131-238 152-162 178-184 217-227 1-115 I079D11 1932 134-241 157-167 183-189 222-230 1-118 I079E06 1933 136-244 158-168 184-190 223-233 1-120 I079E08 1934 137-247 159-171 187-193 226-236 1-121 I079E11 1935 136-243 159-169 185-191 224-232 1-120 I079E12 1936 143-253 165-177 193-199 232-242 1-127 I079F01 1937 133-241 154-164 180-186 219-230 1-117 I079F02 1938 148-253 169-179 195-201 234-242 1-132 I079F03 1939 140-247 161-171 187-193 226-236 1-124 I079F04 1940 136-243 157-167 183-189 222-232 1-120 I079F09 1941 130-237 151-161 177-183 216-226 1-114 I079F10 1942 136-243 157-167 183-189 222-232 1-120 I079F12 1943 136-243 159-169 185-191 224-232 1-120 I079G02 1944 136-243 157-167 183-189 222-232 1-120 I079G05 1945 136-243 159-169 185-191 224-232 1-120 I079G06 1946 135-245 157-170 186-192 225-234 1-119 I079H05 1947 136-243 157-167 183-189 222-232 1-120 I079H06 1948 134-241 157-167 183-189 222-230 1-118 I080A01 1949 132-242 154-166 182-188 221-231 1-115 I080A02 1950 134-245 156-169 185-191 224-234 1-117 I080A05 1951 142-253 164-177 193-199 232-242 1-125 I080A06 1952 143-250 166-176 192-198 231-239 1-125 I080A07 1953 136-247 158-171 187-193 226-236 1-119 I080A10 1954 142-252 164-176 192-198 231-241 1-125 I080B02 1955 140-248 162-172 188-194 227-237 1-122 I080B03 1956 139-249 161-173 189-195 228-238 1-122 I080B05 1957 139-249 161-173 189-195 228-238 1-121 I080B06 1958 143-254 165-177 193-199 232-243 1-126 I080B07 1959 135-243 157-167 183-189 222-232 1-117 I080B08 1960 137-248 159-172 188-194 227-237 1-120 I080B09 1961 143-254 165-178 194-200 233-243 1-126 I080B10 1962 139-249 161-173 189-195 228-238 1-122 I080B11 1963 138-248 160-172 188-194 227-237 1-121 I080B12 1964 141-253 164-179 195-201 234-242 1-123 I0800O3 1965 140-248 162-172 188-194 227-237 1-122 I080006 1966 146-254 168-178 194-200 233-243 1-128 I080007 1967 145-256 167-180 196-202 235-245 1-128 I080008 1968 138-249 160-173 189-195 228-238 1-121 I080C10 1969 132-243 154-167 183-189 222-232 1-115 I080C11 1970 138-249 160-173 189-195 228-238 1-121 I080C12 1971 139-249 161-173 189-195 228-238 1-122 I080D01 1972 138-245 161-171 187-193 226-234 1-120 I080D02 1973 142-254 164-177 193-199 232-243 1-125 I080D04 1974 140-248 162-172 188-194 227-237 1-122 I080D05 1975 138-246 160-170 186-192 225-235 1-120 I080D08 1976 138-248 160-172 188-194 227-237 1-121 I080D09 1977 139-250 161-174 190-196 229-239 1-122 I080D11 1978 136-247 158-171 187-193 226-236 1-119 I080D12 1979 137-245 159-169 185-191 224-234 1-119 I080E01 1980 138-246 160-170 186-192 225-235 1-120 I080E04 1981 137-247 159-171 187-193 226-236 1-120 I080E06 1982 138-248 160-172 188-194 227-237 1-121 I080E07 1983 143-254 165-178 194-200 233-243 1-126 I080E08 1984 138-249 160-173 189-195 228-238 1-121 I080E12 1985 132-242 154-166 182-188 221-231 1-114 I080F04 1986 139-249 161-173 189-195 228-238 1-122 I080F05 1987 143-253 165-177 193-199 232-242 1-126 I080F06 1988 140-248 162-172 188-194 227-237 1-122 I080F08 1989 132-240 154-164 180-186 219-229 1-114 I080G03 1990 142-250 164-174 190-196 229-239 1-124 I080G04 1991 133-244 156-171 187-193 226-233 1-115 I080G10 1992 145-252 167-177 193-199 232-241 1-127 I080G11 1993 137-247 159-171 187-193 226-236 1-120 I080H01 1994 142-252 164-176 192-198 231-241 1-124 I080H02 1995 140-248 162-172 188-194 227-237 1-123 I080H03 1996 136-246 158-170 186-192 225-235 1-119 I080H04 1997 138-249 160-173 189-195 228-238 1-121 I080H05 1998 137-247 159-171 187-193 226-236 1-120 I080H06 1999 138-249 160-173 189-195 228-238 1-121 I080H07 2000 138-248 160-172 188-194 227-237 1-121 I080H08 2001 140-251 162-175 191-197 230-240 1-122 I080H09 2002 141-249 163-173 189-195 228-238 1-123 I081A01 2003 130-237 153-163 179-185 218-226 1-114 I081A03 2004 135-245 157-170 186-192 225-234 1-119 I081A04 2005 130-237 153-163 179-185 218-226 1-114 I081A06 2006 130-237 151-161 177-183 216-226 1-114 I081A08 2007 130-240 152-164 180-186 219-229 1-114 I081A09 2008 134-241 155-165 181-187 220-230 1-118 I081A10 2009 133-243 155-168 184-190 223-232 1-117 I081B01 2010 130-236 151-161 177-183 216-225 1-114 I081B04 2011 134-244 156-169 185-191 224-233 1-118 I081B05 2012 133-243 155-168 184-190 223-232 1-117 I081B06 2013 133-240 154-164 180-186 219-229 1-117 I081B07 2014 136-243 157-167 183-189 222-232 1-120 I081B08 2015 132-239 153-163 179-185 218-228 1-116 I081B09 2016 130-240 152-164 180-186 219-229 1-114 I081B10 2017 130-237 153-163 179-185 218-226 1-114 I081B11 2018 132-239 153-163 179-185 218-228 1-116 I081C07 2019 130-237 153-163 179-185 218-226 1-114 I081C08 2020 130-237 153-163 179-185 218-226 1-114 I081D04 2021 135-242 156-166 182-188 221-231 1-119 I081D06 2022 132-239 153-163 179-185 218-228 1-116 I081D08 2023 132-239 153-163 179-185 218-228 1-116 I081D09 2024 130-238 152-162 178-184 217-227 1-114 I081D10 2025 130-240 152-164 180-186 219-229 1-114 I081D11 2026 134-244 156-169 185-191 224-233 1-118 I081D12 2027 130-237 153-163 179-185 218-226 1-114 I081E02 2028 130-237 153-163 179-185 218-226 1-114 I081E03 2029 130-240 152-164 180-186 219-229 1-114 I081E05 2030 130-240 152-164 180-186 219-229 1-114 I081E06 2031 134-241 155-165 181-187 220-230 1-118 I081E07 2032 134-241 155-165 181-187 220-230 1-118 I081E10 2033 142-249 163-173 189-195 228-238 1-126 I081F01 2034 130-239 152-164 180-186 219-228 1-114 I081F04 2035 132-239 153-163 179-185 218-228 1-116 I081F05 2036 130-237 151-161 177-183 216-226 1-114 I081F06 2037 134-244 156-169 185-191 224-233 1-118 I081F07 2038 132-239 153-163 179-185 218-228 1-116 I081F11 2039 130-237 151-161 177-183 216-226 1-114 I081G01 2040 130-237 153-163 179-185 218-226 1-114 I081G04 2041 130-240 152-164 180-186 219-229 1-114 I081G06 2042 135-245 157-170 186-192 225-234 1-119 I081G10 2043 130-237 153-163 179-185 218-226 1-114 I081H02 2044 130-240 152-164 180-186 219-229 1-114 I081H03 2045 130-240 152-164 180-186 219-229 1-114 I081H04 2046 135-242 156-166 182-188 221-231 1-119 I081H06 2047 130-240 152-165 181-187 220-229 1-114 I081H08 2048 130-240 152-164 180-186 219-229 1-114 I082A02 2049 139-249 161-173 189-195 228-238 1-123 I082A04 2050 130-240 152-165 181-187 220-229 1-114 I082A08 2051 134-243 156-168 184-190 223-232 1-118 I082A11 2052 130-240 152-165 181-187 220-229 1-114 I082B06 2053 131-238 154-164 180-186 219-227 1-115 I082B09 2054 134-241 157-167 183-189 222-230 1-118 I082B12 2055 131-241 153-166 182-188 221-230 1-115 I082C01 2056 136-243 157-167 183-189 222-232 1-120 I082C05 2057 136-243 157-167 183-189 222-232 1-120 I082C08 2058 137-244 158-168 184-190 223-233 1-121 I082D02 2059 130-240 152-165 181-187 220-229 1-114 I082E05 2060 134-241 155-165 181-187 220-230 1-118 I082E06 2061 130-240 152-165 181-187 220-229 1-114 I082E07 2062 139-246 162-172 188-194 227-235 1-123 I082F11 2063 136-243 159-169 185-191 224-232 1-120 I082G07 2064 136-243 159-169 185-191 224-232 1-120 I082G10 2065 138-249 160-173 189-195 228-238 1-118 I082G11 2066 143-250 164-174 190-196 229-239 1-127 I082H04 2067 132-238 153-163 179-185 218-227 1-116 I082H09 2068 139-246 160-170 186-192 225-235 1-123 I083A06 2069 137-244 159-169 185-191 224-233 1-120 I083A09 2070 138-248 160-172 188-194 227-237 1-121 I083A11 2071 136-248 158-171 187-193 226-237 1-119 I083B03 2072 139-247 161-171 187-193 226-236 1-121 I083B05 2073 139-250 161-174 190-196 229-239 1-122 I083B06 2074 139-250 161-174 190-196 229-239 1-122 I083B10 2075 139-246 162-172 188-194 227-235 1-121 I083C01 2076 136-247 158-171 187-193 226-236 1-119 I083C02 2077 136-246 158-171 187-193 226-235 1-119 I083C07 2078 137-249 159-172 188-194 227-238 1-120 I083C12 2079 136-246 158-171 187-193 226-235 1-119 I083D04 2080 146-256 168-181 197-203 236-245 1-129 I083D07 2081 150-262 173-188 204-210 243-251 1-132 I083D08 2082 143-254 165-178 194-200 233-243 1-126 I083D10 2083 147-258 169-181 197-203 236-247 1-130 I083D12 2084 134-242 156-166 182-188 221-231 1-116 I083E02 2085 139-249 161-173 189-195 228-238 1-122 I083E03 2086 136-248 158-171 187-193 226-237 1-119 I083E04 2087 144-255 166-179 195-201 234-244 1-127 I083E08 2088 140-248 162-172 188-194 227-237 1-122 I083E12 2089 135-245 157-170 186-192 225-234 1-118 I083F02 2090 146-258 168-181 197-203 236-247 1-129 I083F04 2091 138-248 160-172 188-194 227-237 1-121 I083F06 2092 135-247 157-170 186-192 225-236 1-118 I083F08 2093 139-250 161-174 190-196 229-239 1-122 I083F11 2094 137-248 159-172 188-194 227-237 1-120 I083G04 2095 139-250 161-174 190-196 229-239 1-122 I083G05 2096 139-249 161-173 189-195 228-238 1-121 I083G06 2097 139-250 161-174 190-196 229-239 1-122 I083G08 2098 142-253 164-177 193-199 232-242 1-125 I083G09 2099 132-242 154-166 182-188 221-231 1-114 I083G11 2100 141-252 163-176 192-198 231-241 1-124 I083H04 2101 142-253 164-177 193-199 232-242 1-125 I083H05 2102 135-243 157-167 183-189 222-232 1-117 I083H07 2103 139-247 161-171 187-193 226-236 1-121 I084A03 2104 130-237 153-163 179-185 218-226 1-114 I084A08 2105 130-240 152-164 180-186 219-229 1-114 I084B08 2106 135-242 156-166 182-188 221-231 1-119 I084C02 2107 136-243 157-167 183-189 222-232 1-120 I084D03 2108 130-240 152-164 180-186 219-229 1-114 I084D05 2109 133-243 155-168 184-190 223-232 1-117 I084E01 2110 130-237 153-163 179-185 218-226 1-114 I084E06 2111 130-237 153-163 179-185 218-226 1-114 I084E10 2112 130-237 151-161 177-183 216-226 1-114 I084E12 2113 130-240 152-164 180-186 219-229 1-114 I084F04 2114 130-237 153-163 179-185 218-226 1-114 I084F07 2115 130-237 153-163 179-185 218-226 1-114 I084F12 2116 135-245 157-170 186-192 225-234 1-119 I084G12 2117 130-240 152-164 180-186 219-229 1-114 I084H02 2118 130-237 153-163 179-185 218-226 1-114 I099B05 2119 146-256 168-180 196-202 235-245 1-129 I099G09 2120 139-249 161-173 189-195 228-238 1-122 I099H01 2121 140-248 162-172 188-194 227-237 1-122 I099H06 2122 139-249 161-173 189-195 228-238 1-122 I099H08 2123 145-255 167-179 195-201 234-244 1-128 I100A01 2124 137-247 159-172 188-194 227-236 1-120 I100A10 2125 141-251 163-175 191-197 230-240 1-124 I100B03 2126 137-247 159-172 188-194 227-236 1-120 I100B04 2127 137-247 159-172 188-194 227-236 1-120 I100C03 2128 141-251 163-175 191-197 230-240 1-124 BAB2001 3240 140-247 163-173 189-195 228-236 1-124 BAB2080 3241 135-245 157-169 185-191 224-234 1-119 BAB2015 3242 140-247 163-173 189-195 228-236 1-124 BAB2019 3243 141-251 163-176 192-198 231-240 1-125 BAB2087 3244 144-253 167-177 193-199 232-242 1-128 BAB2016 3245 141-251 163-176 192-198 231-246 1-125 BAB2034 3246 146-258 170-183 199-205 238-247 1-132 BAB2065 3247 141-250 162-175 191-197 230-239 1-125 AAs of AAs of  AAs of VH Clone ID VH CDR1 VH CDR2 CDR3 VH CDR3 Sequence (SEQ ID NO:) I003F12S 26-35 50-66 99-111 HDDDVLTGYYFES (SEQ ID NO: 2130) I006D08 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I008A11 26-37 52-69 102-117  DRYDILTGYYYYGMDV (SEQ ID NO: 2129) I017D10 26-35 50-66 99-121 VQMDSEYYDLLTGINVGPYYFDY (SEQ ID NO: 2132) I022D01 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I031F02 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I050A12 26-35 50-66 99-113 APYDLLTHYFHYFDY (SEQ ID NO: 2134) I051C04 26-35 50-66 99-118 AATTSQKHNKYAYYFYGMDV (SEQ ID NO: 2131) I050B11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I050B11-01 26-35 50-66 99-114 PFYDTLTSYVFQVWVA (SEQ ID NO: 2143) I050B11-02 26-35 50-66 99-114 PFYDTLTSYVFQVWVA (SEQ ID NO: 2143) I050B11-03 26-35 50-66 99-114 PFYDTLTRYVFQYFDH (SEQ ID NO: 2144) I050B11-04 26-35 50-66 99-114 PFYDTLTGYVFQYFDH (SEQ ID NO: 2141) I050B11-05 26-35 50-66 99-114 PFYDTLTRYVFQVWVA (SEQ ID NO: 2142) I050B11-06 26-35 50-66 99-114 PFYDTLTGYVFQVWVA (SEQ ID NO: 2140) I050B11-07 26-35 50-66 99-114 PFYDTLTRYVFQYFDH (SEQ ID NO: 2144) I050B11-08 26-35 50-66 99-114 PFYDTLTGYVFQYFDH (SEQ ID NO: 2141) I050B11-09 26-35 50-66 99-114 PFYDTLTRYVFQVWVA (SEQ ID NO: 2142) I050B11-10 26-35 50-66 99-114 PFYDTLTRYVFQVWVA (SEQ ID NO: 2142) I050B11-11 26-35 50-66 99-114 PFYDTLTGYVFQVWVA (SEQ ID NO: 2140) I050B11-12 26-35 50-66 99-114 PFYDTLTGYVFQVWVA (SEQ ID NO: 2140) I050B11-13 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I050B11-14 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I050B11-15 26-35 50-66 99-114 PFYDTLTSYVFQVWVA (SEQ ID NO: 2143) I050B11-16 26-35 50-66 99-114 PFYDTLTSYVFQVWVA (SEQ ID NO: 2143) I050B11-17 26-35 50-66 99-114 PFYDTLTRYVFQYFDH (SEQ ID NO: 2144) I050B11-18 26-35 50-66 99-114 PFYDTLTRYVFQYFDH (SEQ ID NO: 2144) I050B11-19 26-35 50-66 99-114 PFYDILTSYVFQYFDH (SEQ ID NO: 2139) I050B11-20 26-35 50-66 99-114 PFYDILTSYVFQYFDH (SEQ ID NO: 2139) I050B11-21 26-35 50-66 99-114 PFYDILTRYVFQYFDH (SEQ ID NO: 2138) I050B11-22 26-35 50-66 99-114 PFYDILTRYVFQYFDH (SEQ ID NO: 2138) I050B11-23 26-35 50-66 99-114 PFYDILTRYVFQYFDH (SEQ ID NO: 2138) I050B11-24 26-35 50-66 99-114 PFYDILTSYVFQYFDH (SEQ ID NO: 2139) I050B11-25 26-35 50-66 99-114 PFYDTLTRYVFQYFDH (SEQ ID NO: 2144) I050B11-26 26-35 50-66 99-114 PFYDILTSYVFQYFDH (SEQ ID NO: 2139) I050B11-27 26-35 50-66 99-114 PFYDILTRYVFQYFDH (SEQ ID NO: 2138) I050B11-28 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093D03 26-35 50-66 99-114 PFYDTLTSYVLGYYLS (SEQ ID NO: 2145) I093D09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093G08 26-35 50-66 99-114 PFYDTLTSYVFQVWVA (SEQ ID NO: 2143) I097D11 26-35 50-66 99-114 PFYDILTSYVFQYFDH (SEQ ID NO: 2139) I101A04 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I101B01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I102A02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I102E01 26-35 50-66 99-114 PFYDTLTRYVFQYFDH (SEQ ID NO: 2144) I102G06 26-35 50-66 99-114 PFYDTLTGYVFQYFDH (SEQ ID NO: 2141) I087A07 26-35 50-66 99-114 PFYDTLTSYVLPRVIP (SEQ ID NO: 2227) I087A08 26-35 50-66 99-113 PFYDTLTSYVCRPHF (SEQ ID NO: 2238) I087A09 26-35 50-66 99-113 PFYDTLTSYVRCPYV (SEQ ID NO: 2272) I087B02 26-35 50-66 99-113 PFYDTLTSYVFRPDL (SEQ ID NO: 2281) I087B03 26-35 50-66 99-113 PFYDTLTSYVKSMPT (SEQ ID NO: 2305) I087B04 26-35 50-66 99-113 PFYDTLTSYVPFLYC (SEQ ID NO: 2292) I087B05 26-35 50-66 99-113 PFYDTLTSYVPVPST (SEQ ID NO: 2270) I087B06 26-35 50-66 99-113 PFYDTLTSYVGIHGL (SEQ ID NO: 2282) I087B08 26-35 50-66 99-114 PFYDTLTSYVPCSPPR (SEQ ID NO: 2261) I087B09 26-35 50-66 99-113 PFYDTLTSYVCYPPA (SEQ ID NO: 2240) I087C02 26-35 50-66 99-113 PFYDTLTSYVLPLLS (SEQ ID NO: 2224) I087C05 26-35 50-66 99-113 PFYDTLTSYVALYRL (SEQ ID NO: 2234) I087C06 26-35 50-66 99-113 PFYDTLTSYVRASFS (SEQ ID NO: 2271) I087C07 26-35 50-66 99-113 PFYDTLTSYVCTPVP (SEQ ID NO: 2319) I087C08 26-35 50-66 99-114 PFYDTLTSYVWPSFFS (SEQ ID NO: 2277) I087D01 26-35 50-66 99-113 PFYDTLTSYVTPRGY (SEQ ID NO: 2275) I087D02 26-35 50-66 99-113 PFYDTLTSYVSSLLS (SEQ ID NO: 2213) I087D03 26-35 50-66 99-114 PFYDTLTSYVPLLPLC (SEQ ID NO: 2263) I087D05 26-35 50-66 99-114 PFYDTLTSYVPPPSFL (SEQ ID NO: 2266) I087D07 26-35 50-66 99-113 PFYDTLTSYVPTSTT (SEQ ID NO: 2269) I087D09 26-35 50-66 99-114 PFYDTLTSYVISCSWA (SEQ ID NO: 2299) I087E04 26-35 50-66 99-114 PFYDTLTSYVSALPPP (SEQ ID NO: 2274) I087E05 26-35 50-66 99-113 PFYDTLTSYVCRHLF (SEQ ID NO: 2236) I087E10 26-35 50-66 99-114 PFYDTLTSYVVSFPSL (SEQ ID NO: 2307) I087F02 26-35 50-66 99-114 PFYDTLTSYVMGVTPS (SEQ ID NO: 2322) I087F04 26-35 50-66 99-114 PFYDTLTSYVLFRPVL (SEQ ID NO: 2326) I087F05 26-35 50-66 99-113 PFYDTLTSYVPSVGG (SEQ ID NO: 2267) I087F07 26-35 50-66 99-113 PFYDTLTSYVPPTRH (SEQ ID NO: 2286) I087F08 26-35 50-66 99-113 PFYDTLTSYVLRSRD (SEQ ID NO: 2243) I087F09 26-35 50-66 99-113 PFYDTLTSYVPLLPP (SEQ ID NO: 2310) I087G05 26-35 50-66 99-113 PFYDTLTSYVLRCVL (SEQ ID NO: 2239) I087G06 26-35 50-66 99-113 PFYDTLTSYVHPSRS (SEQ ID NO: 2285) I087G07 26-35 50-66 99-114 PFYDTLTSYVLRLPPQ (SEQ ID NO: 2241) I087G09 26-35 50-66 99-113 PFYDTLTSYVGPYGT (SEQ ID NO: 2284) I087G10 26-35 50-66 99-113 PFYDTLTSYVTTPCT (SEQ ID NO: 2276) I087H02 26-35 50-66 99-110 ASYLSTSSSLDN (SEQ ID NO: 2265) I088A01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088A03 26-35 50-66 99-114 PFYDTLTSYVIPFLPL (SEQ ID NO: 2290) I088A04 26-35 50-66 99-114 PFYDTLTSYVLHIYPH (SEQ ID NO: 2335) I088A08 26-35 50-66 99-114 PFYDTLTNYVFEYYAS (SEQ ID NO: 2323) I088A09 26-35 50-66 99-114 PFYDTLTSYVILYYLH (SEQ ID NO: 2295) I088A10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088A11 26-35 50-66 99-114 PFYDTLTSYVLMYFPH (SEQ ID NO: 2220) I088A12 26-35 50-66 99-114 PFYDTLTSYVLFFYPL (SEQ ID NO: 2325) I088B01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088B02 26-35 50-66 99-114 PFYDTLTSYVFDYYAS (SEQ ID NO: 2244) I088B03 26-35 50-66 99-114 PFYDTLTSYVIPFLPL (SEQ ID NO: 2290) I088B05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088B06 26-35 50-66 99-114 PFYDTLTSYVFEYYSL (SEQ ID NO: 2324) I088B07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088B08 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088B09 26-35 50-66 99-114 PFYDTLTSYVLEFYLL (SEQ ID NO: 2303) I088B10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088B12 26-35 50-66 99-113 PFYDTLTSYVLPLDS (SEQ ID NO: 2223) I088C01 26-35 50-66 99-114 PFYDTLTSYVLYFYPS (SEQ ID NO: 2317) I088C03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088C09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088C12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088D01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088D03 26-35 50-66 99-114 PFYDTLTSYVLHYYAL (SEQ ID NO: 2215) I088D04 26-35 50-66 99-113 PFYDTLTSYVLPPSV (SEQ ID NO: 2225) I088D07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088D08 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088D11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088E01 23-32 47-63 96-111 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088E02 26-35 50-66 99-114 PFYDTLTSYVLHYYLY (SEQ ID NO: 2216) I088E03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088E04 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088E08 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088E10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088E11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088F07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088G02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088G03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088G07 26-35 50-66 99-114 PFYDTLTSYVFHYYPL (SEQ ID NO: 2260) I088G09 26-35 50-66 99-114 PFYDTLTSYVFPVYYL (SEQ ID NO: 2264) I088G10 26-35 50-66 99-114 PFYDTLTSYVLHFIDH (SEQ ID NO: 2301) I088H05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I088H07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092A03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092A05 26-35 50-66 99-114 PFYDTLTSYVFHYYDV (SEQ ID NO: 2258) I092A06 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092A08 26-35 50-66 99-114 PFYDTLTSYVHEFFSL (SEQ ID NO: 2283) I092A10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092A11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092B01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092B02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092B04 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092B05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092B10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092B12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092C01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092C02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092C07 26-35 50-66 99-113 PFYDTLTSYVLALDL (SEQ ID NO: 2328) I092C08 26-35 50-66 99-114 PFYDTLTSYVFGYYSL (SEQ ID NO: 2254) I092C12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092D01 26-35 50-66 99-114 PFYDTLTSYVLKYYTD (SEQ ID NO: 2226) I092D07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092D09 26-35 50-66 99-114 PFYDTLTSYVMHAYPL (SEQ ID NO: 2255) I092D10 26-35 50-66 99-114 PFYDTLTSYVFHYLPV (SEQ ID NO: 2256) I092D11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092E01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092E03 26-35 50-66 99-114 PFYDTLTSYAFQYFDH (SEQ ID NO: 2230) I092E04 26-35 50-66 99-114 PFYDTLTSYVFEYFSV (SEQ ID NO: 2248) I092E07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092E10 26-35 50-66 99-114 PFYDTLTSYVLFYYPL (SEQ ID NO: 2327) I092E11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092F01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092F02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092F05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092F07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092F08 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092F11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092F12 26-35 50-66 99-114 PFYDTLTSYVLAYYPD (SEQ ID NO: 2306) I092G01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092G05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092G10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I092H01 26-35 50-66 99-110 ASYLSTSSSLDN (SEQ ID NO: 2265) I093A06 26-35 50-66 99-114 PFYDTLTSYVLPVYDH (SEQ ID NO: 2334) I093A09 26-35 50-66 99-114 PFYDTLTSYVFQYFAH (SEQ ID NO: 2268) I093A11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093A12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093B02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093B05 26-35 50-66 99-114 PFYDTLTSYVIFYYPT (SEQ ID NO: 2289) I093B06 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093B09 26-35 50-66 99-114 PFYDTLTSYVLEVYHP (SEQ ID NO: 2318) I093B12 26-35 50-66 99-114 PFYDTLTSYVFAPLVT (SEQ ID NO: 2242) I093C02 26-35 50-66 99-114 PFYDTLTSYVLHAYAF (SEQ ID NO: 2332) I093C03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093C05 26-35 50-66 99-114 PFYDTLTSYVILYYLH (SEQ ID NO: 2295) I093D05 26-35 50-66 99-114 PFYDTLTSYVFEFLPL (SEQ ID NO: 2245) I093D08 26-35 50-66 99-114 PFYDTLTSYVRPFYAH (SEQ ID NO: 2273) I093D10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093D12 26-35 50-66 99-114 PFYDTLTSYVLHFYRV (SEQ ID NO: 2302) I093E01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093E02 26-35 50-66 99-114 PFYDTLTSYVIQYFDH (SEQ ID NO: 2297) I093E05 26-35 50-66 99-114 PFYDTLTSYVHEFFSL (SEQ ID NO: 2283) I093E08 26-35 50-66 99-114 PFYDTLTSYVMQFFPT (SEQ ID NO: 2321) I093E10 26-35 50-66 99-114 PFYDTLTSYVLSFYPV (SEQ ID NO: 2246) I093F01 26-35 50-66 99-114 PFYDTLTSYVLYYYAF (SEQ ID NO: 2251) I093F03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093F05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093F08 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093F11 26-35 50-66 99-114 PFYDTLTSYVLHFYPL (SEQ ID NO: 2333) I093G07 26-35 50-66 99-114 PFYDTLTSYVLQYYVL (SEQ ID NO: 2237) I093G11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093G12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I093H06 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094A08 26-35 50-66 99-114 PFYDTLTSYVFQYFDY (SEQ ID NO: 2280) I094B07 26-35 50-66 99-114 PFYDTLTSYVLPVWVS (SEQ ID NO: 2228) I094B08 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094B12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094C11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094C12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094D06 26-35 50-66 99-114 PFYDTLTSYVIEYYPV (SEQ ID NO: 2288) I094D07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094D08 26-35 50-66 99-114 PFYDTLTSYVLHYLPL (SEQ ID NO: 2314) I094D09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094D10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094D11 26-35 50-66 99-114 PFYDTLTSYVFHFYPV (SEQ ID NO: 2218) I094E04 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094E08 26-35 50-66 99-114 PFYDTLTSYVLEAFSL (SEQ ID NO: 2311) I094F04 26-35 50-66 99-114 PFYDTLTSYVFGFYPF (SEQ ID NO: 2252) I094F05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094F10 26-35 50-66 99-114 PIYDTLTSYVFQYFDH (SEQ ID NO: 2278) I094F11 26-35 50-66 99-114 PFYDTLTSYVLWYYQD (SEQ ID NO: 2249) I094F12 26-35 50-66 99-114 PFYDTLTSYVIPFYPL (SEQ ID NO: 2296) I094G06 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I094G10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I095A04 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I095A12 26-35 50-66 99-114 PFYDTLTSYVLEYFPL (SEQ ID NO: 2320) I095B04 26-35 50-66 99-114 PFYDTLTSYVLEFFPA (SEQ ID NO: 2312) I095B09 26-35 50-66 99-114 PFYDTLTSYVIEYLPL (SEQ ID NO: 2287) I095B10 26-35 50-66 99-114 PFYDTLTSYVLHYYSA (SEQ ID NO: 2217) I095C02 26-35 50-66 99-114 PFYDTLTSYVLFYYTA (SEQ ID NO: 2331) I095C05 26-35 50-66 99-114 PFYDTLTSYVLHYLPV (SEQ ID NO: 2337) I095C07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I095C08 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I095C09 26-35 50-66 99-114 PFYDTLTSYVMHYYPT (SEQ ID NO: 2259) I095D01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I095D02 26-35 50-66 99-114 PFYDTLTSYVLQYFRY (SEQ ID NO: 2235) I095D03 26-35 50-66 99-114 PFYDTLTSYVLQVFDT (SEQ ID NO: 2233) I095D05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I095D09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I095E01 26-35 50-66 99-114 PFYDTLTSYVLDYYSS (SEQ ID NO: 2309) I095E05 26-35 50-66 99-114 PFYDALTSYVFQYFDH (SEQ ID NO: 2221) I095E12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I095F06 26-35 50-66 99-114 PFYDTLTSYVFPFYPH (SEQ ID NO: 2262) I095F09 26-35 50-66 99-114 PFYDTLTSYVIGFYPV (SEQ ID NO: 2291) I095G06 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I095G09 26-35 50-66 99-114 PFYDTLTSYVMDFYSV (SEQ ID NO: 2253) I095G11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096A01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096A10 26-35 50-66 99-114 PFYDTLTSYVLPFYAL (SEQ ID NO: 2222) I096B01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096B03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096C01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096C06 26-35 50-66 99-114 PFYDTLTSYVLPYLTH (SEQ ID NO: 2229) I096C09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096D01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096D02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096D05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096D06 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096D09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096E02 26-35 50-66 99-114 PFYDTLTSYVLGFYPV (SEQ ID NO: 2329) I096E06 26-35 50-66 99-114 PFYDTLTSYVLHYHTH (SEQ ID NO: 2336) I096E11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096F02 26-35 50-66 99-114 PFYDTLTSYVIHFLPL (SEQ ID NO: 2330) I096G01 26-35 50-66 99-114 PFYDTLTSYVIPFLPL (SEQ ID NO: 2290) I096G02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096G05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096G07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I096G09 26-35 50-66 99-114 PFYDTLTSYVMHYLPV (SEQ ID NO: 2257) I096G12 26-35 50-66 99-114 PFYDTLTSYVLEFFSH (SEQ ID NO: 2315) I096H01 26-35 50-66 99-110 ASYLSTSSSLDN (SEQ ID NO: 2265) I097A04 26-35 50-66 99-114 PFYDTLTSYVIHYLVT (SEQ ID NO: 2294) I097A06 26-35 50-66 99-114 PFYDTLTSYVLPYYTL (SEQ ID NO: 2231) I097A09 26-35 50-66 99-114 PFYDTLTSYVLHYYPI (SEQ ID NO: 2298) I097B02 26-35 50-66 99-114 PFYDTLTSYVLWFYPL (SEQ ID NO: 2247) I097B09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I097B10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I097B11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I097C05 26-35 50-66 99-114 PFYDTLTSYVLHYYTH (SEQ ID NO: 2219) I097C09 26-35 50-66 99-114 PFYDTLTSYVLHYYAY (SEQ ID NO: 2316) I097C11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I097D05 26-35 50-66 99-114 PFYDTLTSYVIHFYSL (SEQ ID NO: 2293) I097D06 26-35 50-66 99-114 PFYDTLTSYVFGFFPH (SEQ ID NO: 2300) I097E01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I097E04 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I097E08 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I097E09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I097F09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I097G10 26-35 50-66 99-114 PFYDTLTSYVFQYFAH (SEQ ID NO: 2268) I097H02 26-35 50-66 99-110 ASYLSTSSSLDN (SEQ ID NO: 2265) I098A04 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I098A05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I098B08 26-35 50-66 99-114 PFYDTLTSYVLDFYSV (SEQ ID NO: 2308) I098C01 26-35 50-66 99-114 PIYDTLTSYVFQYFDH (SEQ ID NO: 2278) I098C04 26-35 50-66 99-114 PFYDTLTSYVLYYYAF (SEQ ID NO: 2251) I098F11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I098F12 26-35 50-66 99-114 PFYDTLTSYVFFFYPF (SEQ ID NO: 2250) I098G02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I098G12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I098H05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I101A01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I101B04 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I101B06 26-35 50-66 99-114 PFYDTLTSYVIPFLTH (SEQ ID NO: 2304) I101D04 26-35 50-66 99-114 PFYDTLTSYVLEFFPD (SEQ ID NO: 2313) I101D07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I101E09 26-35 50-66 99-114 PFYDTLTSYVFQYFDR (SEQ ID NO: 2279) I101E12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I101G02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I101G11 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I102C03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I102E09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I102F02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I102G08 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I102G09 26-35 50-66 99-114 PFYDTLTSYVLHYYAH (SEQ ID NO: 2214) I106A09 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I106B02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I106B06 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I106C07 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I106E05 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I106E12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I106G01 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I106G03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I109B06 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I109D12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I109E12 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I109G06 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I109H04 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I110B03 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I112D09 26-35 50-66 99-114 PFYDTLTSYGFQYFDH (SEQ ID NO: 2232) I112F10 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I089F12 26-35 50-66 99-112 SRDLLLFPHHGLDS (SEQ ID NO: 2146) I105E12 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I108D08 26-35 50-66 99-112 SRDLLLFPLAPLYP (SEQ ID NO: 2148) I108E06 26-35 50-66 99-112 SRDLLLFPHHGLDV (SEQ ID NO: 2151) I113E07 26-35 50-66 99-112 SRDLLLFPHHSLDL (SEQ ID NO: 2152) I114G05 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I116A01 26-35 50-66 99-112 SRDLLLFPHHALSP (SEQ ID NO: 2149) I116A09 26-35 50-66 99-112 SRYLLLFPHHSFDL (SEQ ID NO: 2150) I116C11 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I085A01 26-35 50-66 99-112 SRDLLLFPHDHLLF (SEQ ID NO: 2602) I085A02 26-35 50-66 99-112 SRDLLLFPSDPLGF (SEQ ID NO: 2639) I085A03 26-35 50-66 99-112 SRDLLLFPTHPLSF (SEQ ID NO: 2561) I085A04 26-35 50-66 99-112 SRDLLLFPLAPLFF (SEQ ID NO: 2550) I085A05 26-35 50-66 99-112 SRDLLLFPSDPLSL (SEQ ID NO: 2659) I085A06 26-35 50-66 99-112 SRDLLLFPSAPLSF (SEQ ID NO: 2611) I085A07 26-35 50-66 99-112 SRDLLLFPASPLSF (SEQ ID NO: 2390) I085A09 26-35 50-66 99-111 SRDLLLFPNDALS (SEQ ID NO: 2632) I085A10 26-35 50-66 99-112 SRDLLLFPSAPLRF (SEQ ID NO: 2609) I085A11 26-35 50-66 99-111 SRDLLLFPHDPLE (SEQ ID NO: 2363) I085B01 26-35 50-66 99-112 SRDLLLFPQSPLYP (SEQ ID NO: 2466) I085B02 26-35 50-66 99-112 SRDLLLFPHSSLVF (SEQ ID NO: 2392) I085B03 26-35 50-66 99-112 SRDLLLFPYDPLLF (SEQ ID NO: 2638) I085B04 26-35 50-66 99-112 SRDLLLFPHAPLYF (SEQ ID NO: 2589) I085B05 26-35 50-66 99-112 SRDLLLFPHAPLSP (SEQ ID NO: 2573) I085B06 26-35 50-66 99-112 SRDLLLFPLSPLSF (SEQ ID NO: 2574) I085B07 26-35 50-66 99-112 SRDLLLFPDFPMAP (SEQ ID NO: 2433) I085B10 26-35 50-66 99-111 SRDLLLFPHSPLY (SEQ ID NO: 2470) I085B12 26-35 50-66 99-112 SRDLLLFPQDPLSP (SEQ ID NO: 2372) I085C02 26-35 50-66 99-112 SRDLLLFPDDPLLS (SEQ ID NO: 2430) I085C03 26-35 50-66 99-112 SRDLLLFPHGPLLI (SEQ ID NO: 2400) I085C05 26-35 50-66 99-112 SRDLLLFPGSPLLF (SEQ ID NO: 2491) I085C06 26-35 50-66 99-112 SRDLLLFPTAALSF (SEQ ID NO: 2341) I085C07 26-35 50-66 99-112 SRDLLLFPHTPLRF (SEQ ID NO: 2375) I085C09 26-35 50-66 99-111 SRDLLLFPHSPLT (SEQ ID NO: 2468) I085C10 26-35 50-66 99-112 SRDLLLFPFSPLLF (SEQ ID NO: 2471) I085C12 26-35 50-66 99-112 SRDLLLFPSHPLFF (SEQ ID NO: 2680) I085D01 26-35 50-66 99-112 SRDLLLFPRPLLLF (SEQ ID NO: 2548) I085D02 26-35 50-66 99-112 SRDLLLFPSQYLDF (SEQ ID NO: 2523) I085D03 26-35 50-66 99-112 SRDLLLFPSSPLLF (SEQ ID NO: 2713) I085D04 26-35 50-66 99-112 SRDLLLFPYFPLVF (SEQ ID NO: 2646) I085D06 26-35 50-66 99-112 SRDLLLFPGSPLLD (SEQ ID NO: 2488) I085D07 26-35 50-66 99-112 SRDLLLFPQAPLLF (SEQ ID NO: 2694) I085D08 26-35 50-66 99-112 SRDLLLFPHSYLSP (SEQ ID NO: 2477) I085D09 26-35 50-66 99-112 SRDLLLFPQTPLFP (SEQ ID NO: 2467) I085D10 26-35 50-66 99-112 SRDLLLFPHSPLHP (SEQ ID NO: 2563) I085D11 26-35 50-66 99-112 SRDLLLFPHAPLAP (SEQ ID NO: 2510) I085D12 26-35 50-66 99-112 SRDLLLFPHTTLRF (SEQ ID NO: 2495) I085E01 26-35 50-66 99-112 SRDLLLFPYAVLHF (SEQ ID NO: 2620) I085E02 26-35 50-66 99-112 SRDLLLFPTSPLRL (SEQ ID NO: 2575) I085E07 26-35 50-66 99-112 SRDLLLFPSDALSF (SEQ ID NO: 2568) I085E08 26-35 50-66 99-112 SRDLLLFPNAPLDP (SEQ ID NO: 2603) I085E09 26-35 50-66 99-112 SRDLLLFPHDPPRF (SEQ ID NO: 2628) I085E10 26-35 50-66 99-112 SRDLLLFPSEPLWP (SEQ ID NO: 2668) I085E11 26-35 50-66 99-112 SRDLLLFPSSPLSN (SEQ ID NO: 2716) I085E12 26-35 50-66 99-112 SRDLLLFPHLPLTP (SEQ ID NO: 2431) I085F01 26-35 50-66 99-112 SRDLLLFPRSPLLF (SEQ ID NO: 2551) I085F02 26-35 50-66 99-112 SRDLLLFPTSPLQL (SEQ ID NO: 2376) I085F03 26-35 50-66 99-112 SRDLLLFPYTPLLF (SEQ ID NO: 2682) I085F04 26-35 50-66 99-112 SRDLLLFPSSPLAF (SEQ ID NO: 2707) I085F05 26-35 50-66 99-112 SRDLLLFPHDPLYF (SEQ ID NO: 2706) I085F06 26-35 50-66 99-112 SRDLLLFPSAHLLF (SEQ ID NO: 2586) I085F07 26-35 50-66 99-112 SRDLLLFPAGPLRF (SEQ ID NO: 2410) I085F09 26-35 50-66 99-112 SRDLLLFPDHAFFV (SEQ ID NO: 2439) I085F10 26-35 50-66 99-112 SRDLLLFPSDSGFA (SEQ ID NO: 2662) I085F11 26-35 50-66 99-112 SRDLLLFPSSYLEF (SEQ ID NO: 2339) I085F12 26-35 50-66 99-112 SRDLLLFPRDPLII (SEQ ID NO: 2558) I085G01 26-35 50-66 99-112 SRDLLLFPSAPLHP (SEQ ID NO: 2605) I085G02 26-35 50-66 99-112 SRDLLLFPNAPLLL (SEQ ID NO: 2613) I085G03 26-35 50-66 99-112 SRDLLLFPAAPLLF (SEQ ID NO: 2403) I085G04 26-35 50-66 99-112 SRDLLLFPSAPLDP (SEQ ID NO: 2601) I085G07 26-35 50-66 99-112 SRDLLLFPNAVLDI (SEQ ID NO: 2629) I085G08 26-35 50-66 99-112 SRDLLLFPSEPLFF (SEQ ID NO: 2664) I085G09 26-35 50-66 99-112 SRDLLLFPSSVLWP (SEQ ID NO: 2338) I085G10 26-35 50-66 99-111 SRDLLLFPHAPLQ (SEQ ID NO: 2554) I085G11 26-35 50-66 99-112 SRDLLLFPDSPLAP (SEQ ID NO: 2445) I085G12 26-35 50-66 99-112 SRDLLLFPSSPLHP (SEQ ID NO: 2576) I085H10 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I086A03 26-35 50-66 99-112 SRDLLLFPSMPLTF (SEQ ID NO: 2695) I086A04 26-35 50-66 99-112 SRDLLLFPHSILHP (SEQ ID NO: 2438) I086A05 26-35 50-66 99-112 SRDLLLFPHAPLSH (SEQ ID NO: 2569) I086A07 26-35 50-66 99-112 SRDLLLFPDAALRF (SEQ ID NO: 2421) I086A09 26-35 50-66 99-112 SRDLLLFPSSHLSF (SEQ ID NO: 2704) I086A10 26-35 50-66 99-112 SRDLLLFPSAPLSS (SEQ ID NO: 2624) I086A11 26-35 50-66 99-112 SRDLLLFPHAPLTP (SEQ ID NO: 2577) I086A12 26-35 50-66 99-112 SRDLLLFPYDPLHS (SEQ ID NO: 2635) I086B02 26-35 50-66 99-112 SRDLLLFPHFPLHP (SEQ ID NO: 2348) I086B03 26-35 50-66 99-112 SRDLLLFPAHPLLF (SEQ ID NO: 2412) I086B05 26-35 50-66 99-112 SRDLLLFPFEPLII (SEQ ID NO: 2457) I086B06 26-35 50-66 99-112 SRDLLLFPASPLNP (SEQ ID NO: 2364) I086B07 26-35 50-66 99-112 SRDLLLFPSSPLYF (SEQ ID NO: 2720) I086B09 26-35 50-66 99-112 SRDLLLFPTSPLSF (SEQ ID NO: 2579) I086B10 26-35 50-66 99-112 SRDLLLFPDDGLSS (SEQ ID NO: 2428) I086B11 26-35 50-66 99-112 SRDLLLFPISPLCF (SEQ ID NO: 2530) I086C03 26-35 50-66 99-112 SRDLLLFPTAPLYG (SEQ ID NO: 2535) I086C05 26-35 50-66 99-112 SRDLLLFPHHSLFF (SEQ ID NO: 2427) I086C07 26-35 50-66 99-112 SRDLLLFPQGPLRF (SEQ ID NO: 2440) I086C08 26-35 50-66 99-112 SRDLLLFPAAPLAF (SEQ ID NO: 2401) I086C09 26-35 50-66 99-112 SRDLLLFPHLPLLF (SEQ ID NO: 2350) I086C10 26-35 50-66 99-112 SRDLLLFPTFPLIF (SEQ ID NO: 2541) I086C11 26-35 50-66 99-112 SRDLLLFPDDPLSF (SEQ ID NO: 2432) I086C12 26-35 50-66 99-112 SRDLLLFPTDSLLF (SEQ ID NO: 2622) I086D01 26-35 50-66 99-112 SRDLLLFPSAPLTP (SEQ ID NO: 2630) I086D04 26-35 50-66 99-112 SRYLLLFPYAPLYD (SEQ ID NO: 2697) I086D05 26-35 50-66 99-112 SRDLLLFPHSPLSF (SEQ ID NO: 2461) I086D06 26-35 50-66 99-112 SRDLLLFPTAPLDL (SEQ ID NO: 2379) I086D07 26-35 50-66 99-112 SRDLLLFPHTHLTF (SEQ ID NO: 2365) I086D08 26-35 50-66 99-112 SRDLLLFPHSSLDF (SEQ ID NO: 2473) I086D09 26-35 50-66 99-112 SRDLLLFPNHPMFP (SEQ ID NO: 2665) I086D10 26-35 50-66 99-112 SRDLLLFPLSSLEF (SEQ ID NO: 2587) I086D11 26-35 50-66 99-112 SRDLLLFPNAPLHP (SEQ ID NO: 2610) I086D12 26-35 50-66 99-112 SRDLLLFPRAHLRF (SEQ ID NO: 2469) I086E02 26-35 50-66 99-112 SRDLLLFPYDPLHF (SEQ ID NO: 2621) I086E03 26-35 50-66 99-112 SRDLLLFPHDALQS (SEQ ID NO: 2598) I086E05 26-35 50-66 99-112 SRDLLLFPRTPLTF (SEQ ID NO: 2567) I086E07 26-35 50-66 99-112 SRDLLLFPAAHLSF (SEQ ID NO: 2398) I086E08 26-35 50-66 99-112 SRDLLLFPRAPLLF (SEQ ID NO: 2490) I086E09 26-35 50-66 99-112 SRDLLLFPFSPLAP (SEQ ID NO: 2464) I086E10 26-35 50-66 99-112 SRDLLLFPRAPLDF (SEQ ID NO: 2367) I086E12 26-35 50-66 99-112 SRDLLLFPTAPLRF (SEQ ID NO: 2522) I086F02 26-35 50-66 99-112 SRDLLLFPSSPLRI (SEQ ID NO: 2714) I086F05 26-35 50-66 99-112 SRDLLLFPTEPLQF (SEQ ID NO: 2540) I086F08 26-35 50-66 99-112 SRDLLLFPSDPLSA (SEQ ID NO: 2643) I086F09 26-35 50-66 99-112 SRDLLLFPYNPPIF (SEQ ID NO: 2653) I086F11 26-35 50-66 99-112 SRDLLLFPHTPLLF (SEQ ID NO: 2489) I086G03 26-35 50-66 99-112 SRDLLLFPHAPLDL (SEQ ID NO: 2513) I086G04 26-35 50-66 99-112 SRDLLLFPFDPLLI (SEQ ID NO: 2454) I086G05 26-35 50-66 99-112 SRDLLLFPTDALRI (SEQ ID NO: 2537) I086G06 26-35 50-66 99-112 SRDLLLFPAAPLTP (SEQ ID NO: 2407) I086G07 26-35 50-66 99-112 SRDLLLFPEGPLLF (SEQ ID NO: 2448) I086G09 26-35 50-66 99-112 SRDLLLFPYAPLSF (SEQ ID NO: 2385) I086G10 26-35 50-66 99-112 SRDLLLFPADSLSF (SEQ ID NO: 2391) I086H05 26-35 50-66 99-112 SRDLLLFPYSPLTH (SEQ ID NO: 2679) I089A01 26-35 50-66 99-111 SRDLLLFPHDPLI (SEQ ID NO: 2612) I089A03 26-35 50-66 99-112 SRDLLLFPLTPLLI (SEQ ID NO: 2590) I089A06 26-35 50-66 99-112 SRDLLLFPHTPLHF (SEQ ID NO: 2485) I089A07 26-35 50-66 99-112 SRDLLLFPTDALYF (SEQ ID NO: 2539) I089A08 26-35 50-66 99-112 SRDLLLFPYTPLLF (SEQ ID NO: 2682) I089A10 26-35 50-66 99-112 SRDLLLFPHQPLTF (SEQ ID NO: 2436) I089A11 26-35 50-66 99-112 SRDLLLFPRTYLDF (SEQ ID NO: 2572) I089B01 26-35 50-66 99-112 SRDLLLFPHSPLHS (SEQ ID NO: 2450) I089B02 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I089B03 26-35 50-66 99-112 SRDLLLFPTSPLQP (SEQ ID NO: 2528) I089B04 26-35 50-66 99-112 SRDLLLFPTHPLLF (SEQ ID NO: 2556) I089B05 26-35 50-66 99-112 SRDLLLFPSSPLIF (SEQ ID NO: 2712) I089B06 26-35 50-66 99-112 SRDLLLFPMAPLSP (SEQ ID NO: 2596) I089B07 26-35 50-66 99-112 SRDLLLFPYSGLDA (SEQ ID NO: 2374) I089B08 26-35 50-66 99-112 SRDLLLFPAAPLSP (SEQ ID NO: 2405) I089B09 26-35 50-66 99-112 SRDLLLFPKSPILF (SEQ ID NO: 2384) I089B10 26-35 50-66 99-112 SRDLLLFPTSPLFF (SEQ ID NO: 2571) I089B11 26-35 50-66 99-112 SRDLLLFPNSPLFP (SEQ ID NO: 2388) I089C01 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I089C02 26-35 50-66 99-112 SRDLLLFPRSPLLF (SEQ ID NO: 2551) I089C03 26-35 50-66 99-112 SRDLLLFPYHPLLF (SEQ ID NO: 2532) I089C05 26-35 50-66 99-112 SRDLLLFPSSALRF (SEQ ID NO: 2722) I089C06 26-35 50-66 99-112 SRDLLLFPSPYLSF (SEQ ID NO: 2701) I089C07 26-35 50-66 99-112 SRDLLLFPQAPLFD (SEQ ID NO: 2683) I089C09 26-35 50-66 99-112 SRDLLLFPHAPFTF (SEQ ID NO: 2507) I089D01 26-35 50-66 99-112 SRDLLLFPHAPLVL (SEQ ID NO: 2581) I089D02 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I089D03 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I089D04 26-35 50-66 99-112 SRDLLLFPSSPLSP (SEQ ID NO: 2717) I089D05 26-35 50-66 99-112 SRDLLLFPHAPLFT (SEQ ID NO: 2546) I089D07 26-35 50-66 99-112 SRDLLLFPNDPLLI (SEQ ID NO: 2634) I089D08 26-35 50-66 99-111 SRDLLLFPHAPLQ (SEQ ID NO: 2554) I089D09 26-35 50-66 99-112 SRDLLLFPSHAFHE (SEQ ID NO: 2677) I089D11 26-35 50-66 99-112 SRDLLLFPNHPLYP (SEQ ID NO: 2663) I089E01 26-35 50-66 99-112 SRDLLLFPYSPLFP (SEQ ID NO: 2657) I089E02 26-35 50-66 99-112 SRDLLLFPQDPLHP (SEQ ID NO: 2346) I089E03 26-35 50-66 99-112 SRDLLLFPDAPLFP (SEQ ID NO: 2423) I089E04 26-35 50-66 99-112 SRDLLLFPHSPLLI (SEQ ID NO: 2453) I089E06 26-35 50-66 99-112 SRDLLLFPGSPLLF (SEQ ID NO: 2491) I089E09 26-35 50-66 99-112 SRDLLLFPSSPLTF (SEQ ID NO: 2718) I089E10 26-35 50-66 99-112 SRDLLLFPTQPLSF (SEQ ID NO: 2566) I089E11 26-35 50-66 99-112 SRDLLLFPLSPLWP (SEQ ID NO: 2578) I089F01 26-35 50-66 99-112 SRDLLLFPTFPLLF (SEQ ID NO: 2380) I089F03 26-35 50-66 99-112 SRDLLLFPHDPLLL (SEQ ID NO: 2580) I089F04 26-35 50-66 99-112 SRDLLLFPYSPLLF (SEQ ID NO: 2670) I089F05 26-35 50-66 99-112 SRDLLLFPHSPLRI (SEQ ID NO: 2459) I089F06 26-35 50-66 99-112 SRDLLLFPRAPLLF (SEQ ID NO: 2490) I089F08 26-35 50-66 99-112 SRDLLLFPRTPLTF (SEQ ID NO: 2567) I089F09 26-35 50-66 99-112 SRDLLLFPLAPLSF (SEQ ID NO: 2555) I089F10 26-35 50-66 99-112 SRDLLLFPNQPLSF (SEQ ID NO: 2667) I089F11 26-35 50-66 99-112 SRDLLLFPLEPMHF (SEQ ID NO: 2565) I089G01 26-35 50-66 99-112 SRDLLLFPSAPLTF (SEQ ID NO: 2626) I089G02 26-35 50-66 99-112 SRDLLLFPSHPLLF (SEQ ID NO: 2687) I089G03 26-35 50-66 99-112 SRDLLLFPRTPLVF (SEQ ID NO: 2721) I089G05 26-35 50-66 99-112 SRDLLLFPGSPLTF (SEQ ID NO: 2389) I089G06 26-35 50-66 99-112 SRDLLLFPTAPLLF (SEQ ID NO: 2514) I089G07 26-35 50-66 99-112 SRDLLLFPSAPLDF (SEQ ID NO: 2597) I089G08 26-35 50-66 99-112 SRDLLLFPSHPLSF (SEQ ID NO: 2688) I089G11 26-35 50-66 99-112 SRDLLLFPSFPLLF (SEQ ID NO: 2671) I089H10 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I090A02 26-35 50-66 99-112 SRDLLLFPAKPLLF (SEQ ID NO: 2416) I090A03 26-35 50-66 99-112 SRDLLLFPNSTLSF (SEQ ID NO: 2678) I090A04 26-35 50-66 99-112 SRDLLLFPDAPLTP (SEQ ID NO: 2426) I090A05 26-35 50-66 99-112 SRDLLLFPHEPLLI (SEQ ID NO: 2648) I090A06 26-35 50-66 99-112 SRDLLLFPTYPLSF (SEQ ID NO: 2600) I090A07 26-35 50-66 99-112 SRDLLLFPTEPLVL (SEQ ID NO: 2479) I090A08 26-35 50-66 99-112 SRDLLLFPTYPLHF (SEQ ID NO: 2584) I090B01 26-35 50-66 99-112 SRDLLLFPHDPLTF (SEQ ID NO: 2627) I090B03 26-35 50-66 99-112 SRDLLLFPQAPLTN (SEQ ID NO: 2705) I090B04 26-35 50-66 99-112 SRDLLLFPHAPLEA (SEQ ID NO: 2520) I090B05 26-35 50-66 99-112 SRDLLLFPDHPLLF (SEQ ID NO: 2442) I090B06 26-35 50-66 99-112 SRDLLLFPRAPLSF (SEQ ID NO: 2496) I090B08 26-35 50-66 99-112 SRDLLLFPRGPLRF (SEQ ID NO: 2542) I090B11 26-35 50-66 99-112 SRDLLLFPFTPLTF (SEQ ID NO: 2474) I090B12 26-35 50-66 99-112 SRDLLLFPQHPLSP (SEQ ID NO: 2452) I090001 26-35 50-66 99-112 SRDLLLFPSAPIVF (SEQ ID NO: 2591) I0900O2 26-35 50-66 99-112 SRDLLLFPQAPLTF (SEQ ID NO: 2702) I0900O3 26-35 50-66 99-112 SRDLLLFPRAPLRF (SEQ ID NO: 2493) I090005 26-35 50-66 99-112 SRDLLLFPRTPLTF (SEQ ID NO: 2567) I090006 26-35 50-66 99-112 SRDLLLFPHAPLDF (SEQ ID NO: 2538) I090007 26-35 50-66 99-112 SRDLLLFPHAGFDS (SEQ ID NO: 2498) I090008 26-35 50-66 99-112 SRDLLLFPYSPLSF (SEQ ID NO: 2676) I090C10 26-35 50-66 99-112 SRDLLLFPGRPLTF (SEQ ID NO: 2358) I090D02 26-35 50-66 99-112 SRDLLLFPAEHLLF (SEQ ID NO: 2408) I090D03 26-35 50-66 99-112 SRDLLLFPTAPLHP (SEQ ID NO: 2351) I090D04 26-35 50-66 99-112 SRDLLLFPHEPLTA (SEQ ID NO: 2654) I090D05 26-35 50-66 99-112 SRDLLLFPHAPLFE (SEQ ID NO: 2529) I090D06 26-35 50-66 99-112 SRDLLLFPRAPLDF (SEQ ID NO: 2367) I090D07 26-35 50-66 99-112 SRDLLLFPFGTLRF (SEQ ID NO: 2462) I090D08 26-35 50-66 99-112 SRDLLLFPSSPLVF (SEQ ID NO: 2723) I090D09 26-35 50-66 99-112 SRDLLLFPRDPLAF (SEQ ID NO: 2505) I090D12 26-35 50-66 99-112 SRDLLLFPTSPLSF (SEQ ID NO: 2579) I090E04 26-35 50-66 99-112 SRDLLLFPHAPLLL (SEQ ID NO: 2552) I090E05 26-35 50-66 99-112 SRDLLLFPSAPISF (SEQ ID NO: 2588) I090E06 26-35 50-66 99-112 SRDLLLFPQGPLSF (SEQ ID NO: 2443) I090E07 26-35 50-66 99-112 SRDLLLFPGSPLHP (SEQ ID NO: 2484) I090E09 26-35 50-66 99-112 SRDLLLFPSDPLSF (SEQ ID NO: 2647) I090E11 26-35 50-66 99-112 SRDLLLFPHDGLAP (SEQ ID NO: 2700) I090E12 26-35 50-66 99-112 SRDLLLFPTSPLTF (SEQ ID NO: 2582) I090F01 26-35 50-66 99-112 SRDLLLFPNGPLHP (SEQ ID NO: 2649) I090F02 26-35 50-66 99-112 SRDLLLFPQAPLSF (SEQ ID NO: 2696) I090F03 26-35 50-66 99-112 SRDLLLFPTAPLSF (SEQ ID NO: 2526) I090F04 26-35 50-66 99-112 SRDLLLFPFFPLQF (SEQ ID NO: 2460) I090F05 26-35 50-66 99-112 SRDLLLFPLDPLHF (SEQ ID NO: 2359) I090F06 26-35 50-66 99-112 SRDLLLFPSEPLQL (SEQ ID NO: 2666) I090F07 26-35 50-66 99-112 SRDLLLFPFAPLRF (SEQ ID NO: 2451) I090F08 26-35 50-66 99-112 SRDLLLFPLHPLIF (SEQ ID NO: 2570) I090F09 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I090F10 26-35 50-66 99-112 SRDLLLFPRDPLRI (SEQ ID NO: 2527) I090F11 26-35 50-66 99-112 SRDLLLFPSNPLTF (SEQ ID NO: 2698) I090G01 26-35 50-66 99-112 SRDLLLFPTAPLEI (SEQ ID NO: 2347) I090G02 26-35 50-66 99-112 SRDLLLFPRDPLQF (SEQ ID NO: 2395) I090G04 26-35 50-66 99-112 SRDLLLFPHEPLAF (SEQ ID NO: 2633) I090G05 26-35 50-66 99-112 SRDLLLFPRAPLAF (SEQ ID NO: 2472) I090G06 26-35 50-66 99-112 SRDLLLFPYSPLAF (SEQ ID NO: 2656) I090G07 26-35 50-66 99-112 SRDLLLFPHTPLDS (SEQ ID NO: 2480) I090G08 26-35 50-66 99-112 SRDLLLFPHTPLTF (SEQ ID NO: 2492) I090G09 26-35 50-66 99-112 SRDLLLFPSEPLRI (SEQ ID NO: 2356) I090G10 26-35 50-66 99-112 SRDLLLFPTAPLDF (SEQ ID NO: 2343) I090G12 26-35 50-66 99-112 SRDLLLFPNRGLDL (SEQ ID NO: 2669) I091A02 26-35 50-66 99-112 SRDLLLFPYDPLFM (SEQ ID NO: 2724) I091A03 26-35 50-66 99-112 SRDLLLFPHAPLYP (SEQ ID NO: 2592) I091A06 26-35 50-66 99-112 SRDLLLFPSAPLAF (SEQ ID NO: 2594) I091A11 26-35 50-66 99-112 SRDLLLFPHSPITF (SEQ ID NO: 2441) I091B01 26-35 50-66 99-112 SRDLLLFPRYPLFF (SEQ ID NO: 2585) I091B02 26-35 50-66 99-112 SRDLLLFPYAPLDF (SEQ ID NO: 2361) I091B04 26-35 50-66 99-112 SRDLLLFPRDPLQF (SEQ ID NO: 2395) I091B05 26-35 50-66 99-112 SRDLLLFPHAPLEL (SEQ ID NO: 2475) I091B07 26-35 50-66 99-112 SRDLLLFPSAPLTF (SEQ ID NO: 2626) I091B10 26-35 50-66 99-112 SRDLLLFPTAPLAF (SEQ ID NO: 2342) I091B11 26-35 50-66 99-112 SRDLLLFPHSPLDF (SEQ ID NO: 2444) I091B12 26-35 50-66 99-112 SRDLLLFPSHPLTF (SEQ ID NO: 2690) I091C02 26-35 50-66 99-112 SRDLLLFPAHPLVI (SEQ ID NO: 2414) I091C03 26-35 50-66 99-112 SRDLLLFPQAPLYP (SEQ ID NO: 2378) I091C04 26-35 50-66 99-112 SRDLLLFPTAPLTF (SEQ ID NO: 2531) I091C05 26-35 50-66 99-112 SRDLLLFPTTPLHF (SEQ ID NO: 2583) I091C06 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I091C09 26-35 50-66 99-112 SRDLLLFPHHPLSF (SEQ ID NO: 2415) I091C11 26-35 50-66 99-112 SRDLLLFPYHSYDI (SEQ ID NO: 2650) I091C12 26-35 50-66 99-112 SRDLLLFPYATLSF (SEQ ID NO: 2618) I091D01 26-35 50-66 99-112 SRDLLLFPNSPLAP (SEQ ID NO: 2672) I091D02 26-35 50-66 99-112 SRDLLLFPYSPLQP (SEQ ID NO: 2673) I091D04 26-35 50-66 99-112 SRDLLLFPQGPLSF (SEQ ID NO: 2443) I091D05 26-35 50-66 99-112 SRDLLLFPHDPLAP (SEQ ID NO: 2606) I091D06 26-35 50-66 99-111 SRDLLLFPHSPLL (SEQ ID NO: 2456) I091D07 26-35 50-66 99-112 SRDLLLFPNGALRF (SEQ ID NO: 2645) I091D09 26-35 50-66 99-112 SRDLLLFPYSPLRF (SEQ ID NO: 2719) I091E01 26-35 50-66 99-112 SRDLLLFPDAPLHP (SEQ ID NO: 2425) I091E02 26-35 50-66 99-112 SRDLLLFPQAPLFP (SEQ ID NO: 2689) I091E03 26-35 50-66 99-112 SRDLLLFPSAPLWP (SEQ ID NO: 2352) I091E04 26-35 50-66 99-112 SRDLLLFPKSPLAF (SEQ ID NO: 2547) I091E06 26-35 50-66 99-112 SRDLLLFPSSPLHP (SEQ ID NO: 2576) I091E07 26-35 50-66 99-112 SRDLLLFPNHPLTF (SEQ ID NO: 2661) I091E08 26-35 50-66 99-112 SRDLLLFPNAPLDS (SEQ ID NO: 2607) I091E09 26-35 50-66 99-112 SRDLLLFPYAPLDF (SEQ ID NO: 2361) I091E10 26-35 50-66 99-112 SRDLLLFPSSPLEF (SEQ ID NO: 2711) I091F01 26-35 50-66 99-112 SRDLLLFPRAPLFF (SEQ ID NO: 2486) I091F03 26-35 50-66 99-112 SRDLLLFPMAPLVG (SEQ ID NO: 2599) I091F05 26-35 50-66 99-112 SRDLLLFPLAPLHP (SEQ ID NO: 2553) I091F06 26-35 50-66 99-112 SRDLLLFPHDPLGF (SEQ ID NO: 2353) I091F07 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I091F08 26-35 50-66 99-112 SRDLLLFPQSPLLF (SEQ ID NO: 2458) I091F09 26-35 50-66 99-112 SRDLLLFPHEHLSF (SEQ ID NO: 2354) I091F10 26-35 50-66 99-112 SRDLLLFPHSPLDF (SEQ ID NO: 2444) I091F11 26-35 50-66 99-112 SRDLLLFPHSPLSP (SEQ ID NO: 2549) I091F12 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I091G01 26-35 50-66 99-112 SRDLLLFPNAALYP (SEQ ID NO: 2386) I091G03 26-35 50-66 99-112 SRDLLLFPNDPLFG (SEQ ID NO: 2355) I091G04 26-35 50-66 99-112 SRDLLLFPGAPLSP (SEQ ID NO: 2478) I091G05 26-35 50-66 99-112 ARDLLLFPAAPLWP (SEQ ID NO: 2397) I091G06 26-35 50-66 99-111 SRDLLLFPNDPLR (SEQ ID NO: 2637) I091G07 26-35 50-66 99-112 SRDLLLFPTAPLDP (SEQ ID NO: 2345) I091G09 26-35 50-66 99-112 SRDLLLFPTAPLFP (SEQ ID NO: 2349) I091G10 26-35 50-66 99-112 SRDLLLFPSDPLVF (SEQ ID NO: 2660) I091G11 26-35 50-66 99-112 SRDLLLFPGSPLTF (SEQ ID NO: 2389) I091G12 26-35 50-66 99-112 SRDLLLFPYSHLEF (SEQ ID NO: 2655) I104A01 26-35 50-66 99-112 SRDLLLFPQSPLHP (SEQ ID NO: 2455) I104A07 26-35 50-66 99-112 SRDLLLFPQAPLFP (SEQ ID NO: 2689) I104A08 26-35 50-66 99-112 SRDLLLFPYAPLTF (SEQ ID NO: 2617) I104A09 26-35 50-66 99-112 SRDLLLFPQNPLHP (SEQ ID NO: 2506) I104A10 26-35 50-66 99-112 SRDLLLFPHEPLCF (SEQ ID NO: 2636) I104A11 26-35 50-66 99-112 SRDLLLFPSAPLSF (SEQ ID NO: 2611) I104A12 26-35 50-66 99-112 SRDLLLFPMAPLRF (SEQ ID NO: 2593) I104B02 26-35 50-66 99-112 SRDLLLFPRSPLSF (SEQ ID NO: 2557) I104B04 26-35 50-66 99-112 SRDLLLFPSAPLYP (SEQ ID NO: 2387) I104B09 26-35 50-66 99-112 SRDLLLFPRDPLQF (SEQ ID NO: 2395) I104B11 26-35 50-66 99-112 SRDLLLFPTAPLTF (SEQ ID NO: 2531) I104C01 26-35 50-66 99-112 SRDLLLFPYSPLYP (SEQ ID NO: 2710) I104C04 26-35 50-66 99-112 SRDLLLFPASPLIF (SEQ ID NO: 2417) I104C05 26-35 50-66 99-112 SRDLLLFPRHPLLF (SEQ ID NO: 2543) I104C06 26-35 50-66 99-111 SRDLLLFPHAPLE (SEQ ID NO: 2524) I104C07 26-35 50-66 99-112 SRDLLLFPHAPLHP (SEQ ID NO: 2370) I104C09 26-35 50-66 99-112 SRDLLLFPHFPLIF (SEQ ID NO: 2399) I104C11 26-35 50-66 99-112 SRDLLLFPHEPLIF (SEQ ID NO: 2644) I104D01 26-35 50-66 99-112 SRDLLLFPNHAFDL (SEQ ID NO: 2652) I104D02 26-35 50-66 99-112 SRDLLLFPHTILYP (SEQ ID NO: 2497) I104D03 26-35 50-66 99-112 SRDLLLFPDWPLYP (SEQ ID NO: 2483) I104D04 26-35 50-66 99-112 SRDLLLFPHYPLFL (SEQ ID NO: 2511) I104D07 26-35 50-66 99-112 SRDLLLFPQAPLHP (SEQ ID NO: 2691) I104D08 26-35 50-66 99-112 SRDLLLFPHAPMDP (SEQ ID NO: 2595) I104D09 26-35 50-66 99-112 SRDLLLFPRAPLTF (SEQ ID NO: 2500) I104E01 26-35 50-66 99-112 SRDLLLFPRATLEF (SEQ ID NO: 2502) I104E02 26-35 50-66 99-112 SRDLLLFPHSPLFP (SEQ ID NO: 2447) I104E03 26-35 50-66 99-112 SRDLLLFPNDPLVL (SEQ ID NO: 2641) I104E05 26-35 50-66 99-112 SRDLLLFPHDPLYI (SEQ ID NO: 2463) I104E11 26-35 50-66 99-112 SRDLLLFPYAPLSF (SEQ ID NO: 2385) I104E12 26-35 50-66 99-112 SRDLLLFPASPLNP (SEQ ID NO: 2364) I104F02 26-35 50-66 99-112 SRDLLLFPHDPLSP (SEQ ID NO: 2616) I104F03 26-35 50-66 99-112 SRDLLLFPRDPLRF (SEQ ID NO: 2360) I104F04 26-35 50-66 99-112 SRDLLLFPGDPLDF (SEQ ID NO: 2481) I104F05 26-35 50-66 99-112 SRDLLLFPHGPLTF (SEQ ID NO: 2402) I104F06 26-35 50-66 99-112 SRDLLLFPHAPLSP (SEQ ID NO: 2573) I104F07 26-35 50-66 99-112 SRDLLLFPSSPLIL (SEQ ID NO: 2465) I104F10 26-35 50-66 99-112 SRDLLLFPNSPLSP (SEQ ID NO: 2362) I104F11 26-35 50-66 99-112 SRDLLLFPQDPLVF (SEQ ID NO: 2708) I104F12 26-35 50-66 99-112 SRDLLLFPKAPLVF (SEQ ID NO: 2544) I104G04 26-35 50-66 99-112 SRDLLLFPHAPLRF (SEQ ID NO: 2559) I104G05 26-35 50-66 99-112 SRDLLLFPRAPLAP (SEQ ID NO: 2476) I104G09 26-35 50-66 99-112 SRDLLLFPTAPLNF (SEQ ID NO: 2518) I104G11 26-35 50-66 99-112 SRHLLLFPQGPLSF (SEQ ID NO: 2482) I105A02 26-35 50-66 99-112 SRDLLLFPHLPLNP (SEQ ID NO: 2494) I105A03 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I105A04 26-35 50-66 99-112 SRDLLLFPGAPLAP (SEQ ID NO: 2487) I105A08 26-35 50-66 99-112 SRDLLLFPQAPLYP (SEQ ID NO: 2378) I105A09 26-35 50-66 99-112 SRDLLLFPRSPLSF (SEQ ID NO: 2557) I105A11 26-35 50-66 99-112 SRDLLLFPSHSFDI (SEQ ID NO: 2692) I105B04 26-35 50-66 99-112 SRDLLLFPYSPLHP (SEQ ID NO: 2658) I105B05 26-35 50-66 99-112 SRDLLLFPYSPLSF (SEQ ID NO: 2676) I105B07 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I105B08 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I105B10 26-35 50-66 99-112 SRDLLLFPASPLNP (SEQ ID NO: 2364) I105B11 26-35 50-66 99-112 SRDLLLFPHEPLSP (SEQ ID NO: 2651) I105B12 26-35 50-66 99-112 SRDLLLFPLDPLII (SEQ ID NO: 2560) I105C02 26-35 50-66 99-112 SRDLLLFPRAPLAF (SEQ ID NO: 2472) I105C03 26-35 50-66 99-112 SRDLLLFPSSPLSF (SEQ ID NO: 2715) I105C05 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I105C06 26-35 50-66 99-112 SRDLLLFPRAPLDF (SEQ ID NO: 2367) I105C08 26-35 50-66 99-112 SRDLLLFPRSPLTF (SEQ ID NO: 2562) I105C12 26-35 50-66 99-112 SRDLLLFPQHGFDA (SEQ ID NO: 2446) I105D04 26-35 50-66 99-112 SRDLLLFPRDPLRF (SEQ ID NO: 2360) I105D06 26-35 50-66 99-112 SRDLLLFPRDPLSF (SEQ ID NO: 2368) I105D08 26-35 50-66 99-112 SRDLLLFPYAPLAF (SEQ ID NO: 2608) I105D09 26-35 50-66 99-112 SRDLLLFPHAAFDV (SEQ ID NO: 2619) I105D10 26-35 50-66 99-112 SRDLLLFPHEPLFP (SEQ ID NO: 2640) I105D11 26-35 50-66 99-112 SRDLLLFPRSALTF (SEQ ID NO: 2519) I105E01 26-35 50-66 99-112 SRDLLLFPHHSFDS (SEQ ID NO: 2422) I105E06 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I105E11 26-35 50-66 99-112 SRDLLLFPNSPLHP (SEQ ID NO: 2675) I105F03 26-35 50-66 99-112 SRDLLLFPHHPLDS (SEQ ID NO: 2409) I105F06 26-35 50-66 99-112 SRDLLLFPQAPLHP (SEQ ID NO: 2691) I105F07 26-35 50-66 99-112 SRDLLLFPSWPLTF (SEQ ID NO: 2340) I105F09 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I105F12 26-35 50-66 99-112 SRDLLLFPTYPLVF (SEQ ID NO: 2604) I105G03 26-35 50-66 99-112 SRDLLLFPHAPLHP (SEQ ID NO: 2370) I105G08 26-35 50-66 99-112 SRDLLLFPKHPLVF (SEQ ID NO: 2366) I105G09 26-35 50-66 99-112 SRDLLLFPASPLNP (SEQ ID NO: 2364) I105G10 26-35 50-66 99-112 SRDLLLFPHHSFDA (SEQ ID NO: 2419) I105G11 26-35 50-66 99-112 SRDLLLFPHDPLLF (SEQ ID NO: 2614) I107A01 26-35 50-66 99-112 SRDLLLFPRHPLVF (SEQ ID NO: 2545) I107A03 26-35 50-66 99-112 SRDLLLFPRAPLYP (SEQ ID NO: 2501) I107A06 26-35 50-66 99-112 SRDLLLFPHAPLDP (SEQ ID NO: 2369) I107A07 26-35 50-66 99-112 SRDLLLFPNAPLSP (SEQ ID NO: 2371) I107A09 26-35 50-66 99-112 SRDLLLFPQAPLSP (SEQ ID NO: 2699) I107A12 26-35 50-66 99-112 SRDLLLFPHAPLSF (SEQ ID NO: 2564) I107B02 26-35 50-66 99-112 SRDLLLFPHAPLFP (SEQ ID NO: 2533) I107B04 26-35 50-66 99-112 SRDLLLFPASPLTF (SEQ ID NO: 2420) I107B05 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I107C01 24-33 48-64 97-110 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I107C02 26-35 50-66 99-112 SRDLLLFPHYGMYV (SEQ ID NO: 2504) I107C04 26-35 50-66 99-112 SRDLLLFPHYPLHP (SEQ ID NO: 2357) I107C06 26-35 50-66 99-112 SRDLLLFPHAPLAP (SEQ ID NO: 2510) I107C08 26-35 50-66 99-112 SRDLLLFPQAPLEP (SEQ ID NO: 2681) I107C10 26-35 50-66 99-112 SRDLLLFPSHAFDL (SEQ ID NO: 2674) I107D01 26-35 50-66 99-112 SRDLLLFPYAPLDF (SEQ ID NO: 2361) I107D04 26-35 50-66 99-112 SRDLLLFPNAPLSF (SEQ ID NO: 2625) I107D07 26-35 50-66 99-112 SRDLLLFPSHSFDV (SEQ ID NO: 2693) I107D12 26-35 50-66 99-112 SRDLLLFPHHSFDT (SEQ ID NO: 2424) I107E01 26-35 50-66 99-112 SRDLLLFPMLGLDL (SEQ ID NO: 2499) I107E05 26-35 50-66 99-112 SRDLLLFPRAPLDF (SEQ ID NO: 2367) I107E07 26-35 50-66 99-112 SRDLLLFPRSPLLF (SEQ ID NO: 2551) I107E09 26-35 50-66 99-112 SRDLLLFPKAPLTF (SEQ ID NO: 2382) I107F01 26-35 50-66 99-112 SRDLLLFPSAPLSP (SEQ ID NO: 2623) I107F05 26-35 50-66 99-112 SRDLLLFPHAPLAP (SEQ ID NO: 2510) I107F09 26-35 50-66 99-112 SRDLLLFPSAPLAP (SEQ ID NO: 2394) I107F10 26-35 50-66 99-112 SRDLLLFPRTPLLF (SEQ ID NO: 2373) I107G01 26-35 50-66 99-112 SRDLLLFPNAPLSP (SEQ ID NO: 2371) I107G05 26-35 50-66 99-112 SRDLLLFPSAPLYP (SEQ ID NO: 2387) I107H02 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I107H06 26-35 50-66 99-112 SRDLLLFPRAPLSF (SEQ ID NO: 2496) I107H09 26-35 50-66 99-112 SRDLLLFPHYPLEM (SEQ ID NO: 2536) I107H10 26-35 50-66 99-112 SRDLLLFPHAPLAP (SEQ ID NO: 2510) I108A12 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I108B03 26-35 50-66 99-112 SRDLLLFPRDPLLF (SEQ ID NO: 2515) I108B04 26-35 50-66 99-112 SRDLLLFPLSPLVP (SEQ ID NO: 2396) I108C09 26-35 50-66 99-112 SRDLLLFPHDPLGF (SEQ ID NO: 2353) I108C11 26-35 50-66 99-112 SRDLLLFPHHSLLF (SEQ ID NO: 2429) I108D10 26-35 50-66 99-112 SRDLLLFPASPLNP (SEQ ID NO: 2364) I108D11 26-35 50-66 99-112 SRDLLLFPASPLNP (SEQ ID NO: 2364) I108D12 26-35 50-66 99-112 SRDLLLFPSAPLNP (SEQ ID NO: 2709) I108E01 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I108E03 26-35 50-66 99-112 SRDLLLFPKHPLRF (SEQ ID NO: 2393) I108E05 26-35 50-66 99-112 SRDLLLFPHAPLFP (SEQ ID NO: 2533) I108E07 26-35 50-66 99-112 SRDLLLFPHAPLDP (SEQ ID NO: 2369) I108E08 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I108E09 26-35 50-66 99-112 SRDLLLFPSAPLSP (SEQ ID NO: 2623) I108E10 26-35 50-66 99-112 SRDLLLFPRDPLDL (SEQ ID NO: 2509) I108E11 26-35 50-66 99-112 SRDLLLFPRDPLEF (SEQ ID NO: 2516) I108F10 26-35 50-66 99-112 SRDLLLFPNAPLSP (SEQ ID NO: 2371) I108F12 26-35 50-66 99-112 SRDLLLFPHYPFDA (SEQ ID NO: 2508) I108G01 26-35 50-66 99-112 SRDLLLFPRDPLRF (SEQ ID NO: 2360) I108G02 26-35 50-66 99-112 SRDLLLFPDAPLAP (SEQ ID NO: 2381) I108G07 26-35 50-66 99-112 SRDLLLFPRAPLAP (SEQ ID NO: 2476) I108G10 26-35 50-66 99-112 SRDLLLFPHHSLLF (SEQ ID NO: 2429) I108G11 26-35 50-66 99-112 SRDLLLFPHHPLTF (SEQ ID NO: 2377) I108G12 26-35 50-66 99-112 SRDLLLFPHHPLTF (SEQ ID NO: 2377) I108H01 26-35 50-66 99-112 SRDLLLFPRDPLHF (SEQ ID NO: 2512) I108H02 26-35 50-66 99-112 SRDLLLFPNAPLNP (SEQ ID NO: 2615) I108H06 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I108H08 26-35 50-66 99-112 SRDLLLFPASPLNP (SEQ ID NO: 2364) I111A06 26-35 50-66 99-112 SRDLLLFPQAPLHP (SEQ ID NO: 2691) I111B12 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I111C01 26-35 50-66 99-112 SRDLLLFPQHGLDL (SEQ ID NO: 2449) I111D06 26-35 50-66 99-112 SRDLLLFPRDPLLF (SEQ ID NO: 2515) I111E04 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I111E10 26-35 50-66 99-112 SRDLLLFPQAPLHP (SEQ ID NO: 2691) I111E11 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I111E12 26-35 50-66 99-112 SRYLLLFPHHSFDL (SEQ ID NO: 2150) I111F07 26-35 50-66 99-112 SRDLLLFPRAPLYP (SEQ ID NO: 2501) I111G02 26-35 50-66 99-112 SRDLLLFPKAPLDF (SEQ ID NO: 2534) I111H10 26-35 50-66 99-112 SRYLLLFPQHGFDA (SEQ ID NO: 2703) I113A04 26-35 50-66 99-112 SRDLLLFPSAPLWP (SEQ ID NO: 2352) I113A12 26-35 50-66 99-112 SRDLLLFPQEPLAP (SEQ ID NO: 2434) I113B06 26-35 50-66 99-112 SRDLLLFPHHPLEP (SEQ ID NO: 2411) I113C06 26-35 50-66 99-112 SRDLLLFPHHGFDA (SEQ ID NO: 2406) I113G04 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I113G05 26-35 50-66 99-112 SRDLLLFPHYSLLL (SEQ ID NO: 2517) I113G10 26-35 50-66 99-112 SRDLLLFPHHPLQF (SEQ ID NO: 2413) I113G11 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I113H06 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I113H07 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I113H09 26-35 50-66 99-112 SRDLLLFPHYTLLF (SEQ ID NO: 2525) I114C04 26-35 50-66 99-112 SRDLLLFPHHGFDA (SEQ ID NO: 2406) I114C12 26-35 50-66 99-112 SRDLLLFPQAPLHP (SEQ ID NO: 2691) I114D04 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I114D06 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I114D10 26-35 50-66 99-112 SRDLLLFPHYSLVL (SEQ ID NO: 2521) I114E01 26-35 50-66 99-112 SRDLLLFPQEPLSP (SEQ ID NO: 2435) I114E02 26-35 50-66 99-112 SRDLLLFPQESFSL (SEQ ID NO: 2437) I114E03 26-35 50-66 99-112 SRDLLLFPKAPLTF (SEQ ID NO: 2382) I114E11 26-35 50-66 99-112 SRDLLLFPHDSFFL (SEQ ID NO: 2383) I114H01 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I114H06 26-35 50-66 99-112 SRDLLLFPHHALDV (SEQ ID NO: 2404) I114H09 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I115A02 26-35 50-66 99-112 SRYLLLFPDHSFDL (SEQ ID NO: 2684) I115A07 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I115B10 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I115C05 26-35 50-66 99-112 SRDLLLFPRAPLYP (SEQ ID NO: 2501) I115C06 26-35 50-66 99-112 SRYLLLFPHHSFDL (SEQ ID NO: 2150) I115C08 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I115C12 26-35 50-66 99-112 SRDLLLFPHHSFDT (SEQ ID NO: 2424) I115D07 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I115E09 26-35 50-66 99-112 SRDLLLFPHHRFDL (SEQ ID NO: 2418) I115F06 26-35 50-66 99-112 SRYLLLFPHYGMDV (SEQ ID NO: 2685) I115F07 26-35 50-66 99-112 SRYLLLFPHYPLLF (SEQ ID NO: 2686) I115F12 26-35 50-66 99-112 SRYLLLFPHHSFDL (SEQ ID NO: 2150) I115G04 26-35 50-66 99-112 SRDLLLFPHHRFDL (SEQ ID NO: 2418) I115G05 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I115G08 26-35 50-66 99-112 SRDLLLFPHDSFDL (SEQ ID NO: 2631) I115H04 26-35 50-66 99-112 SRDLLLFPHANLSP (SEQ ID NO: 2503) I115H07 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I115H09 26-35 50-66 99-112 SRDLLLFPHHRFDL (SEQ ID NO: 2418) I116A07 26-35 50-66 99-112 SRDLLLFPYEPLRF (SEQ ID NO: 2642) I116B01 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I116B12 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I116C06 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I116D07 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I116E02 26-35 50-66 99-112 SRDLLLFPHHRFDL (SEQ ID NO: 2418) I116E04 26-35 50-66 99-112 SRDLLLFPHHSFDL (SEQ ID NO: 2147) I116F02 26-35 50-66 99-112 SRYLLLFPHHSFDL (SEQ ID NO: 2150) I116F11 26-35 50-66 99-112 SRDLLLFPHYPLLF (SEQ ID NO: 2344) I116G05 26-35 50-66 99-112 SRDLLLFPQAPLSP (SEQ ID NO: 2699) I001C09 26-35 50-66 99-116 DGSYDILTGYYIDNYMDV (SEQ ID NO: 2154) I006D07 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I007B03 26-35 50-66 99-116 DGSYDILTGYYIDNYMDV (SEQ ID NO: 2154) I007F11 26-35 50-66 99-113 DGIDILLVPAALMDV (SEQ ID NO: 2160) I007H08 26-37 52-69 102-117 DRYDILTGYYYYGMDV (SEQ ID NO: 2129) I008A09 26-35 50-66 99-119 DREAYYDILTGYYLYYYYMDV (SEQ ID NO: 2172) I008B01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008C02 26-37 52-67 100-118  HVRDYDILTGYYRGHYFDY (SEQ ID NO: 2167) I008C03 26-35 50-65 98-116 EGSYDILTGYYVGVGRMDV (SEQ ID NO: 2171) I008C12 26-35 50-68 101-119  FNPTYDILTGYYIGGYFQH (SEQ ID NO: 2155) I012A06 26-37 52-67 100-118  GRWDYDLLTGEHLGYYFDY (SEQ ID NO: 2162) I016E05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016F02 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I016F04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016H07 26-35 50-66 99-114 GYHDPLTSYNYNWFDP (SEQ ID NO: 2163) I018C02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018C10 26-35 50-66 99-116 DGSYDILTGYYIDNYMDV (SEQ ID NO: 2154) I018D07 26-35 50-66 99-116 DGSYDILTGYYIDNYMDV (SEQ ID NO: 2154) I018H08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018H09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I021B05 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I022E02 26-35 50-66 99-114 ATYDPLTGYSFDGLDI (SEQ ID NO: 2157) I026E03 26-35 50-66 99-114 TDYDILTGYPMGYFDP (SEQ ID NO: 2173) I027A07 26-35 50-66 99-117 GGEYDILTGYYFGLGVYDY (SEQ ID NO: 2170) I028A06 26-35 50-66 99-115 GGDYDILTGLYYYGMDV (SEQ ID NO: 2156) I029D07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I029F11 26-35 50-66 99-116 DGSYDILTGYYIDNYMDV (SEQ ID NO: 2154) I031C03 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031C07 26-35 50-66 99-120 SSPPRWYDALTGDSSYHSAMDV (SEQ ID NO: 2169) I031F09 26-35 50-66 99-116 DEGRDLLTGYYWPNFFDS (SEQ ID NO: 2168) I031G08 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I031G10 26-35 50-66 99-120 SSPPKWYDALTGDSSYHSAMDV (SEQ ID NO: 2165) I031G11 26-35 50-66 99-116 DEGRDLLTGYYWPNFFDS (SEQ ID NO: 2168) I037E07 26-35 50-66 99-113 DGIDILLVPAALMDV (SEQ ID NO: 2160) I037E12 26-35 50-66 99-113 DGIDILLVPAALMDV (SEQ ID NO: 2160) I050A07 26-40 55-71 104-118  QDNDPLTGYKLGFDY (SEQ ID NO: 2164) I061D02 26-37 52-69 102-117  DRYDILTGYYYYGMDV (SEQ ID NO: 2129) I061E07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I061H01 26-35 50-68 101-119  FNPTYDILTGYYIGGYFQH (SEQ ID NO: 2155) I001A03 26-35 50-66 99-117 ERHYYDILTGYQTGYGMDV (SEQ ID NO: 2784) I001A07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I001A08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I001A10 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I001A12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I001B02 26-35 50-66 99-110 DRETKVGYGMDV (SEQ ID NO: 2945) I001B07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I001C06 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I001C08 26-35 50-66 99-117 EGSYDILTGYYVGVGRMDV (SEQ ID NO: 2171) I001C12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I001D08 26-35 50-65 98-113 DSYDILTGYRGYYFDY (SEQ ID NO: 2745) I001D12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I001E05 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I001E07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I001G09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I001H05 26-35 50-66 99-117 ERHYYDILTGYQTGYGMDV (SEQ ID NO: 2784) I001H08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I003A01 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I003A06 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I003A07 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I003A10 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I003B03 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I003B04 25-34 49-65 98-111 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I003B09 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I003C01 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I003C02 26-35 50-66 99-114 GDYDILTGYPAECFQI (SEQ ID NO: 2854) I003C03 26-35 50-66 99-114 GDYDILTGYPAECFQI (SEQ ID NO: 2854) I003C12 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I003D04 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I003E05 26-35 50-66 99-114 GDYDILTGYPAECFQI (SEQ ID NO: 2854) I003F01 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I003F02 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I003G01 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I003G05 26-35 50-66 99-116 GSGYDLLTGYFTGSPLDY (SEQ ID NO: 2766) I003G06 26-35 50-66 99-118 DRGGNYDILTGYYFHHGVDV (SEQ ID NO: 2914) I003G11 26-35 50-66 99-117 DAQSYYDILTGYQSYAFDI (SEQ ID NO: 2183) I003H02 26-35 50-66 99-113 DNYDILTGYSRRFDP (SEQ ID NO: 2942) I003H05 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I003H08 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I005A01 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I005A02 26-35 50-66 99-114 EGRDILTGVYYYGLDV (SEQ ID NO: 2893) I005B01 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I005B09 26-35 50-65 98-110 TYYDILTGRFFDI (SEQ ID NO: 2866) I005C01 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I005D02 26-35 50-66 99-114 DLRYDILTGYHDAFDI (SEQ ID NO: 2890) I005D03 26-35 50-66 99-115 GAYYDILTGYYPYGMDV (SEQ ID NO: 2860) I005E01 26-35 50-66 99-115 GTYYDILTGYFHYGMDV (SEQ ID NO: 2774) I005E08 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I005F01 26-35 50-66 99-113 DQHDILTGVYYGMDV (SEQ ID NO: 2921) I005F02 26-35 50-66 99-117 VSPSYDILTGYYLPHAFDV (SEQ ID NO: 2849) I005F04 26-35 50-65 98-110 TYYDILTGRFFDI (SEQ ID NO: 2866) I005F08 26-35 50-66 99-113 PSYDILTGYLYYFDY (SEQ ID NO: 2850) I005G01 26-35 50-66 99-114 DLRYDILTGYHDAFDI (SEQ ID NO: 2890) I005G08 26-35 50-66 99-115 GAYYDILTGYYPYGMDV (SEQ ID NO: 2860) I005H02 26-35 50-66 99-113 GQYYDILTGYNWFDP (SEQ ID NO: 2857) I006B01 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I006C09 26-35 50-66 99-116 GGYSSGWLRGGPYNWFDP (SEQ ID NO: 2967) I006D09 26-35 50-66 99-114 GDYDILTGYYIPLRDY (SEQ ID NO: 2792) I006E01 26-35 50-68 101-116  NLFDVWTLPYYYYMDV (SEQ ID NO: 2965) I006E07 26-35 50-66 99-116 ADYDILTGYSPLTYGMDV (SEQ ID NO: 2762) I006F01 26-35 50-68 101-113  MYYDILTGHNFDY (SEQ ID NO: 2879) I006F02 26-35 50-66 99-115 VSRDILTGNYYYYGMDV (SEQ ID NO: 2817) I006F07 26-35 50-66 99-116 GGYSSGWLRGGPYNWFDP (SEQ ID NO: 2967) I006G01 26-35 50-68 101-119  AGGYYDILTGRDYYYGMDV (SEQ ID NO: 2877) I006G04 26-35 50-66 99-105 RRYALDY (SEQ ID NO: 2920) I006H01 26-35 50-65 98-119 DRGSYDILTGYYTPPHYYGMDV (SEQ ID NO: 2761) I006H02 26-35 50-66 99-116 GGYSSGWLRGGPYNWFDP (SEQ ID NO: 2967) I007A01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I007A08 26-35 50-66 99-114 SHYDILTGLNYWYFDY (SEQ ID NO: 2746) I007A11 26-35 50-66 99-113 ENYDFLTGYYGAFDI (SEQ ID NO: 2772) I007A12 26-35 50-68 101-117  GIYDILTGYHWDGAFDI (SEQ ID NO: 2892) I007B04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I007C04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I007C08 26-35 50-65 98-115 IRLYCYSLTGYYPYGMDD (SEQ ID NO: 2810) I007C12 26-35 50-66 99-113 TNYDILTGYYQGVDY (SEQ ID NO: 2782) I007D07 26-35 50-66 99-113 GQYYDILTGYNWFDP (SEQ ID NO: 2857) I007D08 26-35 50-68 101-117  GIYDILTGYHWDDAFDI (SEQ ID NO: 2872) I007E03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I007E10 26-35 50-66 99-114 DFYDILTGYPLGGMDV (SEQ ID NO: 2741) I007E11 26-35 50-66 99-117 DLPYYDILTGYSLTSGMDV (SEQ ID NO: 2923) I007F06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I007F08 26-35 50-65 98-116 GRRYDILTGYYYYHHGMDV (SEQ ID NO: 2811) I007G07 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I007G09 26-35 50-66 99-115 DSGGDILTGYYMPYFDY (SEQ ID NO: 2847) I007G10 26-35 50-65 98-115 VGLYYDILTGYYPSGMDV (SEQ ID NO: 2805) I007H07 26-35 50-68 101-120  SQAHYDILTGYYLWSYGMDV (SEQ ID NO: 2875) I007H11 26-35 50-66 99-114 ESYDILTGYRHYGMDL (SEQ ID NO: 2891) I008A02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008A05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008A06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008A07 26-35 50-66 99-115 DREYDLLTGYYLHAFDM (SEQ ID NO: 2960) I008A12 26-35 50-66 99-113 ENYDFLTGYYGAFDI (SEQ ID NO: 2772) I008B02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008B04 26-35 50-66 99-116 DGSYDILTGYYIDNYMDV (SEQ ID NO: 2154) I008B05 26-35 50-66 99-114 DHYDILTGLYYYGMDV (SEQ ID NO: 2760) I008B06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008B07 24-33 48-64 97-113 GRRYDILTGYYKGPLDY (SEQ ID NO: 2902) I008B10 26-35 50-66 99-114 AYYDNLTGFLPYGMGV (SEQ ID NO: 2947) I008B11 26-35 50-66 99-117 EGYDILTGYFLDYYHGMDV (SEQ ID NO: 2753) I008C06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008C08 26-35 50-66 99-122 GPRGGPYYDILTGYYLSLSDAFDI (SEQ ID NO: 2729) I008C09 26-35 50-66 99-115 EYYDILTGYRDPYGMDV (SEQ ID NO: 2973) I008D01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008D02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008D03 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I008D04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008D05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008D06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008D07 26-35 50-66 99-117 DRGYYDILTGYYRGHGMDV (SEQ ID NO: 2837) I008D08 26-35 50-66 99-117 DLPYYDILTGYSLTSGMDV (SEQ ID NO: 2923) I008D12 26-35 50-66 99-117 EEGFYDILTGYYGPGYFDY (SEQ ID NO: 2974) I008E01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008E02 20-31 46-63 96-110 EGYDILTGYSKFLDY (SEQ ID NO: 2906) I008E03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008E04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008E08 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I008E09 26-35 50-66 99-116 ERADYDILTGYYFYDMDV (SEQ ID NO: 2833) I008E12 26-37 52-67 100-114  FRYDILTSYYYGMDV (SEQ ID NO: 2734) I008F03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008F06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008F07 26-35 50-65 98-116 GRRYDILTGYYYYHHGMDV (SEQ ID NO: 2811) I008F08 26-35 50-66 99-116 GHYDILTGYDDYYYGMDV (SEQ ID NO: 2844) I008F09 26-35 50-65 98-106 HDILTGFDY (SEQ ID NO: 2904) I008F10 26-35 50-66 99-113 SGYDILTGYLYGMDV (SEQ ID NO: 2934) I008F11 26-35 50-68 101-117  APYDILTGYSDYYGMDV (SEQ ID NO: 2968) I008G02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008G03 26-35 50-66 99-113 GDYDPLTGYSFGVDV (SEQ ID NO: 2941) I008G04 26-35 50-65 98-116 EGSYDILTGYYVGVGRMDV (SEQ ID NO: 2171) I008G05 26-35 50-66 99-117 DGYYDILTGGFYYYYGMDV (SEQ ID NO: 2899) I008G11 26-35 50-66 99-109 AYYDILTGLDY (SEQ ID NO: 2966) I008G12 26-35 50-66 99-116 DQQYDILTGYYIHYGMDV (SEQ ID NO: 2964) I008H02 26-35 50-66 99-114 DQVDLLLMDHNYYMDV (SEQ ID NO: 2918) I008H03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I008H06 26-35 50-65 98-116 EGSYDILTGYYVGVGRMDV (SEQ ID NO: 2171) I008H09 26-35 50-66 99-116 DQQYDILTGYYIHYGMDV (SEQ ID NO: 2964) I008H11 26-35 50-66 99-114 TKYDILTGYYYYYMDV (SEQ ID NO: 2856) I012B03 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012B06 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012B10 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012C03 26-35 50-66 99-115 TDRFGAKDVTSRWGMDV (SEQ ID NO: 2814) I012C06 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012C09 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012D12 26-35 50-66 99-118 DRGGNYDILTGYYFHHGVDV (SEQ ID NO: 2914) I012E07 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012E08 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I012E09 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012F05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012F12 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012G03 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I012G05 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I012G10 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I012H09 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I013A10 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I013A12 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I013B04 26-35 50-66 99-120 SSPPKWYDALTGDSSYHSAMDV (SEQ ID NO: 2165) I013B09 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I013C02 26-35 50-66 99-120 SSPPKWYDALTGDSSYRSAMDV (SEQ ID NO: 2818) I013C04 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I013D02 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I013D03 26-35 50-66 99-120 SSPPKWYDALTGDSSYHSAMDV (SEQ ID NO: 2165) I013D10 26-35 50-66 99-118 GLRHVTLFGTGTRGHFYMDV (SEQ ID NO: 2789) I013E02 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I013E05 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I013E09 26-35 50-66 99-120 SSPPKWYDALTGDSSYHSAMDV (SEQ ID NO: 2165) I013F03 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I013F04 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I013F07 26-35 50-66 99-118 AATTSQKHNKYAYYFYGMDV (SEQ ID NO: 2131) I013F09 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I013F10 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I013H04 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I013H07 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I014A12 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I014C06 26-35 50-66 99-114 GDYDILTGYPAECFQI (SEQ ID NO: 2854) I014C10 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I014C12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I014E06 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I014F02 26-37 52-67 100-114  AGYDLLTGYPFYFDS (SEQ ID NO: 2757) I016A08 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I016A09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016C02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016C03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016C05 26-35 50-66 99-121 VQMDSEYYDLLTGINVGPYYFDY (SEQ ID NO: 2132) I016C09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016C11 26-35 50-66 99-121 VQMDSEYYDLLTGINVGPYYFDY (SEQ ID NO: 2132) I016D10 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016D11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016E03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016E04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016F03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016F11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016G01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016G06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I016G12 26-35 50-66 99-121 VQMDSEYYDLLTGINVGPYYFDY (SEQ ID NO: 2132) I016H10 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I017A06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I017A07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I017A11 25-34 49-65 98-113 ATYDPLTGYSFDGLDI (SEQ ID NO: 2157) I017E12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I017G03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I017G07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I017H01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018A02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018A04 26-35 50-66 99-117 EGSYDILTGYYVGVGRMDV (SEQ ID NO: 2171) I018A05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018A11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018B02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018B08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018C04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018D02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018E06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018E08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018F04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018G06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I018H07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I019E05 26-35 50-66 99-117 ERHYYDILTGYQTGYGMDV (SEQ ID NO: 2784) I019F06 26-35 50-66 99-117 ERHYYDILTGYQTGYGMDV (SEQ ID NO: 2784) I019G12 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I020D01 26-35 50-66 99-110 DRETKVGYGMDV (SEQ ID NO: 2945) I020D05 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I020E10 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I020G12 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I020H06 24-33 48-64 97-116 EGGNYHILTGYYIGNGAFDI (SEQ ID NO: 2896) I020H10 24-33 48-64 97-116 EGENYDILTGYYIGNGAFDI (SEQ ID NO: 2903) I021A11 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I021B01 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I021C11 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I021D12 26-35 50-66 99-110 DRETKVGYGMDV (SEQ ID NO: 2945) I021E10 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I021G02 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I022A08 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I022B01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I022B10 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I022C02 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I022C04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I022C08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I022D06 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I022E08 26-35 50-66 99-115 ASYYDILTGYYKGAFDI (SEQ ID NO: 2855) I022F01 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I022F04 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I022F12 26-35 50-66 99-113 GDYDILTGTYYYIDV (SEQ ID NO: 2859) I022G11 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I023D01 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I023D04 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I024B04 26-35 50-66 99-113 VYYDILTGYNLFFDY (SEQ ID NO: 2177) I024D01 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I024F06 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I024H01 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I024H07 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I025A01 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I025A04 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I025A07 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I025B01 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I025B10 26-35 50-66 99-113 DNYDILTGYSRRFDP (SEQ ID NO: 2942) I025B12 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I025C07 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I025D11 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I025E04 26-35 50-66 99-115 PLGITAVRGAKTDAFGI (SEQ ID NO: 2929) I025E05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I025E07 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I025E10 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I025F01 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I025F08 26-35 50-66 99-110 GGSSQNFYGMDV (SEQ ID NO: 2884) I025G03 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I025G08 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I025H02 26-35 50-65 98-117 AGSGFHDILTGYYKGGYFDY (SEQ ID NO: 2961) I026A01 26-35 50-66 99-114 GDYDILTGYPAECFQI (SEQ ID NO: 2854) I026B01 26-35 50-66 99-116 GSVYDILTGTYYKSGMGV (SEQ ID NO: 2733) I026B06 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I026C06 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I026C10 26-34 49-65 98-111 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I026C11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I026D09 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I026E04 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I026E06 26-35 50-66 99-113 GYDDILTGYIMALDY (SEQ ID NO: 2821) I026E09 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I026F01 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I026F09 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I026F12 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I026G08 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I026G10 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I026G11 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I026H02 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I026H06 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I026H10 26-35 50-66 99-117 GGEYDILTGYYFGLGVYDY (SEQ ID NO: 2170) I027A09 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I027B02 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I027B05 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I027C08 26-34 49-63 96-111 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I027D02 26-35 50-66 99-114 DPFGAVPGYYYYAMDV (SEQ ID NO: 2826) I027E03 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I027E05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I027F04 26-35 50-66 99-117 GPWYDPLFPPSGRHYGLDV (SEQ ID NO: 2793) I027F05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I027F11 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I027G06 26-35 50-66 99-113 DNYDILTGYSRRFDP (SEQ ID NO: 2942) I027G07 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I027H03 26-35 50-66 99-114 GDYDILTGYPAECFQI (SEQ ID NO: 2854) I028A04 26-35 50-66 99-116 DMYYDILTGYYTGLAFDM (SEQ ID NO: 2880) I028A07 26-35 50-66 99-114 VLNYDILTGYYYGMDV (SEQ ID NO: 2832) I028B08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I028B10 26-35 50-68 101-121  DFGYYDILTGYYIGAFYAFDI (SEQ ID NO: 2861) I028C01 26-37 52-69 102-115  GGHTCIIPTCHMGG (SEQ ID NO: 2796) I028C04 26-35 50-66 99-116 DMYYDILTGYYTGLAFDM (SEQ ID NO: 2880) I028C08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I028D04 26-35 50-65 98-113 ATQDILTGYLYSGMDV (SEQ ID NO: 2977) I028D05 26-35 50-66 99-114 EHYDILTGYSLLGMDV (SEQ ID NO: 2907) I028D12 26-35 50-66 99-116 DGYYDILTGYSVYYGMDV (SEQ ID NO: 2938) I028E06 26-35 50-65 98-116 EGSYDILTGYYVGVGRMDV (SEQ ID NO: 2171) I028E07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I028E08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I028F06 26-35 50-66 99-119 DDRRGYYDILTGYYRFGSFDI (SEQ ID NO: 2901) I028F08 26-35 50-66 99-107 DIDIGGDDS (SEQ ID NO: 2954) I028G08 26-35 50-66 99-114 VSGYNSGYFESYDMDV (SEQ ID NO: 2732) I028G09 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I028G10 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I028H02 26-37 52-69 102-115  SGEPCITLACNLGG (SEQ ID NO: 2797) I028H03 26-35 50-66 99-121 DASEYYDILTGYYLATGRNWFDP (SEQ ID NO: 2888) I028H06 26-35 50-66 99-118 DPSPYYDILTGYFLPYYMDV (SEQ ID NO: 2843) I028H09 26-35 50-68 101-113  EIDDILTGYYMDV (SEQ ID NO: 2905) I029A10 26-35 50-65 98-112 MNYDILTGLVNWFDP (SEQ ID NO: 2786) I029A12 26-35 50-68 101-110  RDILTGFYDS (SEQ ID NO: 2933) I029B11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I029C08 26-35 50-66 99-117 EGSYDILTGYYVGVGRMDV (SEQ ID NO: 2171) I029E10 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I029F08 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I029G08 26-35 50-66 99-114 GYYDILTGYQSDAFDI (SEQ ID NO: 2927) I030A02 26-35 50-66 99-115 TERFGAKDVTARWGMDV (SEQ ID NO: 2874) I030A03 26-35 50-66 99-113 ENYDILTGYYNFFDY (SEQ ID NO: 2737) I030A04 26-35 50-66 99-113 RQYDILTGYYGGFDY (SEQ ID NO: 2958) I030A05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030A09 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030A12 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030B06 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030B08 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030B10 26-35 50-66 99-114 ELGHREGGYWYSPYNV (SEQ ID NO: 2838) I0300O3 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030006 26-35 50-68 101-119  DPGNYDILTGYYYYYGMDV (SEQ ID NO: 2935) I030008 26-35 50-66 99-106 SGPGWFDP (SEQ ID NO: 2870) I030009 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030C10 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030C11 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030C12 26-35 50-66 99-106 SGPGWFDP (SEQ ID NO: 2870) I030D07 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030D12 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030E02 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030E05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030E07 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030E08 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030E09 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030E10 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030F02 26-37 52-67 100-114  AGYDLLTGYPFYFDS (SEQ ID NO: 2757) I030F05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030F06 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030F08 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030F09 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030F11 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030F12 26-35 50-66 99-113 DNYDILTGYSRRFDP (SEQ ID NO: 2942) I030G03 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030G07 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I030G09 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030H05 26-35 50-66 99-118 DRGGNYDILTGYYFHHGVDV (SEQ ID NO: 2914) I030H06 26-37 52-69 102-119  ATKSYDILTRMYYYHMDV (SEQ ID NO: 2748) I030H10 26-35 50-66 99-113 DNYDILTGYSRRFDP (SEQ ID NO: 2942) I030H11 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I031A01 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031A03 26-35 50-66 99-114 PYYDPLTAYTFQYFGN (SEQ ID NO: 2806) I031A08 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031A12 26-35 50-66 99-119 GREDTDKVKPWDRYYHYYMDV (SEQ ID NO: 2972) I031B03 26-35 50-68 101-109  GLGHTDSDS (SEQ ID NO: 2959) I031B06 26-35 50-66 99-115 AKGYYYDSSGASDVFDV (SEQ ID NO: 2871) I031B07 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031B08 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I031B09 26-35 50-66 99-120 SNPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2840) I031B11 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031B12 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031C01 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031C02 26-35 50-66 99-114 PFYDTLTSYVFQYFDH (SEQ ID NO: 2137) I031C04 26-35 50-66 99-120 GRKDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2813) I031C08 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031C11 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031D01 26-35 50-66 99-118 AATTSQKHNKYAYYFYGMDV (SEQ ID NO: 2131) I031D04 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031D06 26-35 50-66 99-120 GREDTDKVKLWDRYYHYYYMDV (SEQ ID NO: 2807) I031D08 26-35 50-66 99-117 VRPKLRYFDWLSRHDAFDL (SEQ ID NO: 2820) I031D09 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031D11 26-35 50-66 99-120 SSPPKWYDALTGDSSYHSAMDV (SEQ ID NO: 2165) I031D12 26-35 50-66 99-117 DKAHGEYGRDYYYYYGMDV (SEQ ID NO: 2735) I031E01 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I031E05 26-35 50-66 99-120 SGPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2848) I031E07 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I031E08 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031E09 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031E10 26-35 50-66 99-120 SSPPKWYDALTGDSSYHSAMDV (SEQ ID NO: 2165) I031E11 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I031F01 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031F04 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031F06 26-35 50-66 99-108 DTVRSGGMDV (SEQ ID NO: 2804) I031F10 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031F11 26-35 50-66 99-117 DKAHGEYGRDYYYYYGMDV (SEQ ID NO: 2735) I031F12 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031G01 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031G03 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I031G05 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031G06 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031G07 26-35 50-66 99-120 SSPPKWYDALTGDSSYHSAMGV (SEQ ID NO: 2816) I031G09 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031G12 26-35 50-66 99-118 AATTSQKHNKYAYYFYGMDV (SEQ ID NO: 2131) I031H01 26-35 50-66 99-110 GYDSSAFRAFDI (SEQ ID NO: 2136) I031H02 26-35 50-66 99-115 AKGYYYDSSGASDVFDV (SEQ ID NO: 2871) I031H03 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I031H06 26-35 50-66 99-117 DKAHGEYGRDYYYYYGMDV (SEQ ID NO: 2735) I031H09 26-35 50-66 99-117 DKAHGEYGRDYYYYYGMDV (SEQ ID NO: 2735) I031H10 26-35 50-66 99-116 DRGYTGYDRLVGGYYFDF (SEQ ID NO: 2931) I031H11 26-35 50-66 99-108 DTVRSGGMDV (SEQ ID NO: 2804) I033A08 26-37 52-69 102-117  DRYDILTGYYYYGMDV (SEQ ID NO: 2129) I033B11 26-37 52-69 102-117  DRYDILTGYYYYGMDV (SEQ ID NO: 2129) I033C01 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I033C08 26-35 50-66 99-115 EMGYDILTGYYLNYMDV (SEQ ID NO: 2862) I033D02 26-35 50-66 99-111 GDYDILTGYYMDV (SEQ ID NO: 2781) I033D03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I033D05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I033D11 26-35 50-66 99-113 VKRDILTGYVEGMDV (SEQ ID NO: 2869) I033D12 26-35 50-66 99-117 GGPHYDILTGYYMAVGFDI (SEQ ID NO: 2962) I033E01 26-35 50-66 99-112 DIDARLAALDAFDI (SEQ ID NO: 2794) I033E06 26-35 50-66 99-114 ATHDPLTGYSFDGFDI (SEQ ID NO: 2780) I033E11 26-35 50-66 99-116 HRSRSCSSTSCRNDAFDI (SEQ ID NO: 2770) I033E12 26-35 50-66 99-115 EMGYDILTGYYLNYMDV (SEQ ID NO: 2862) I033F03 26-35 50-66 99-112 EGAADYLNGQYFQD (SEQ ID NO: 2768) I033F08 26-35 50-66 99-118 QKVYYDILTGYNYYYYGMDV (SEQ ID NO: 2767) I033F10 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I033F12 26-35 50-66 99-107 DIDIGGDDS (SEQ ID NO: 2954) I033G01 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I033G03 26-35 50-66 99-115 PQGVTLVRGAETDAFAI (SEQ ID NO: 2925) I033G08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I033H04 25-34 49-65 98-113 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I037A05 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I037B03 26-35 50-66 99-114 SHYDILTRLNYWYFDL (SEQ ID NO: 2950) I037B04 26-35 50-66 99-117 DPGYYDILTGYFHRYGMDV (SEQ ID NO: 2922) I037C04 26-35 50-65 98-115 ENGDYDILTGQTFYGMDV (SEQ ID NO: 2752) I037C06 26-35 50-66 99-114 LYYDILTGYHWDAFDI (SEQ ID NO: 2882) I037C08 26-35 50-66 99-113 DGIDILLVPAALMDV (SEQ ID NO: 2160) I037D11 26-35 50-66 99-109 SQWLEHDVFDI (SEQ ID NO: 2864) I037E06 26-35 50-66 99-117 DRRDYDLLTRYYYYYGMDV (SEQ ID NO: 2928) I037F04 26-35 50-65 98-117 KQRGDYDILTGYQLGYAFDI (SEQ ID NO: 2808) I037G01 26-35 50-66 99-114 SHYDILTRLNYWYFDL (SEQ ID NO: 2950) I037G03 26-35 50-66 99-119 DLGSFYDILTALRLENYGMDV (SEQ ID NO: 2963) I037G10 26-35 50-66 99-113 DYYDILTKLPYGMDV (SEQ ID NO: 2975) I042A07 26-35 50-66 99-117 VSPSYDILTGYYLPHAFDV (SEQ ID NO: 2849) I042A10 26-35 50-65 98-115 GPRYYDILTGYRYNWFDP (SEQ ID NO: 2801) I042B03 26-35 50-66 99-113 DIDDILTGYVLGMDV (SEQ ID NO: 2924) I042B12 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I042D01 26-35 50-66 99-109 QQWLPYDAFDI (SEQ ID NO: 2839) I042D03 26-35 50-68 101-113  AYYDILTGYFFDI (SEQ ID NO: 2873) I042D10 26-35 50-65 98-115 ERADYDILTGYYFYGMDV (SEQ ID NO: 2802) I042E10 26-37 52-69 102-120  ERPYYDILTGYTVTYGMDV (SEQ ID NO: 2798) I042E11 26-35 50-66 99-113 DEYDILTGLLQGMDV (SEQ ID NO: 2883) I042F08 26-37 52-67 100-115  GDYDILTGYPLHAFDI (SEQ ID NO: 2738) I042F12 26-35 50-66 99-113 DGYDILTGYYFGMDV (SEQ ID NO: 2976) I042G08 26-35 50-66 99-114 EHYDILTGYSLLGMDV (SEQ ID NO: 2907) I042G10 26-35 50-66 99-114 SHYDILTGLNYWYFDL (SEQ ID NO: 2166) I042H03 26-35 50-65 98-116 GSLYYDILTGYYIGNAFDI (SEQ ID NO: 2759) I043A03 26-35 50-66 99-117 DGYYDILTGGFYYYYGMDV (SEQ ID NO: 2899) I043B02 26-35 50-65 98-115 GGYYDILTGYLVYYGMDV (SEQ ID NO: 2744) I043B03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I043B06 26-35 50-66 99-116 DQQYDILTGYHIDYYMDV (SEQ ID NO: 2828) I043B07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I043B09 26-35 50-65 98-116 HVRDYDILTGYYRGHHFDY (SEQ ID NO: 2727) I043D11 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I043E05 26-35 50-66 99-116 TESNYDILTGYYWPSMDV (SEQ ID NO: 2940) I043F01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I043F04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I043F12 26-35 50-66 99-116 TESNYDILTGYYWPSMDV (SEQ ID NO: 2940) I043H07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I044A11 26-35 50-68 101-117  APYDILTGYSDYYGMDV (SEQ ID NO: 2968) I044B11 26-35 50-66 99-112 DSDARLAALDAFDI (SEQ ID NO: 2978) I044C09 26-35 50-66 99-113 GQFGVLPNYYYHMDV (SEQ ID NO: 2943) I044C10 26-35 50-66 99-116 DIKRYNSNWPYYDYYMDV (SEQ ID NO: 2726) I044D03 26-35 50-66 99-117 DKQYYDILTGDPVEGGMDV (SEQ ID NO: 2889) I044D09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I044E07 26-35 50-66 99-110 AGSSLVTYGTDV (SEQ ID NO: 2825) I044E11 26-35 50-66 99-116 SDDYDILTGNYVGSLLDY (SEQ ID NO: 2758) I044F07 26-35 50-66 99-120 DGRLSYDILTGYYARDYYGMDV (SEQ ID NO: 2912) I044G02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I044G07 26-35 50-66 99-122 DQNHPIYDILTGYYVPTGPLELKN (SEQ ID NO: 2845) I044H01 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I050A01 26-35 50-66 99-114 DMGYDILTGYYGAFDI (SEQ ID NO: 2946) I050B12 26-35 50-66 99-114 DYYDVLTGFSLDGMDV (SEQ ID NO: 2829) I050006 26-35 50-65 98-113 DHYDVLTGSYLQAFDV (SEQ ID NO: 2728) I050008 26-37 52-67 100-114  GRYDFLTGYLRNFDY (SEQ ID NO: 2731) I050E01 26-35 50-66 99-113 GHYDILTGYYFGFDY (SEQ ID NO: 2886) I050E10 26-35 50-66 99-110 DMKVYYKYALDV (SEQ ID NO: 2823) I050H08 26-35 50-66 99-114 DLRYDILTGYHDAFDI (SEQ ID NO: 2890) I051A04 26-35 50-66 99-120 SSPPKWYDALTGHSSYHSAMDV (SEQ ID NO: 2159) I051A08 26-35 50-66 99-114 HRRARVVVPVPGAMDV (SEQ ID NO: 2930) I051A12 26-35 50-66 99-116 DGSYDILTGYYIDNYMDV (SEQ ID NO: 2154) I051B08 26-36 51-67 100-115  RSMIVVTTAPYDAFDL (SEQ ID NO: 2785) I051C06 26-35 50-66 99-108 DTVRSGGMDV (SEQ ID NO: 2804) I051G12 26-35 50-66 99-116 DGSYDILTGYYIDNYMDV (SEQ ID NO: 2154) I055A05 26-35 50-66 99-106 SGPGWFDP (SEQ ID NO: 2870) I055A11 26-35 50-66 99-106 SGPGWFDP (SEQ ID NO: 2870) I061A03 26-34 49-65 98-113 ELGSSIVGATTGALDM (SEQ ID NO: 2852) I061A04 26-35 50-66 99-114 GDYDILTGYPAECFQI (SEQ ID NO: 2854) I061A08 26-35 50-66 99-113 DNYDILTGYSRRFDP (SEQ ID NO: 2942) I061A09 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I061A10 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I061B07 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I061B09 24-33 48-64 97-116 EGGNYDILTGYYIGNGAFDI (SEQ ID NO: 2158) I061B12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I061C12 26-35 50-66 99-111 TYYDILTGYHFDY (SEQ ID NO: 2788) I061D01 26-35 50-68 101-110  GPGVIGNYDY (SEQ ID NO: 2749) I061D03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I061D04 26-35 50-66 99-113 AVLRYSAGLQGAFDI (SEQ ID NO: 2970) I061D07 26-35 50-66 99-114 VSGYNSGYFESYDMDV (SEQ ID NO: 2732) I061D09 26-35 50-66 99-114 LNLEKTVVRGFGYFDL (SEQ ID NO: 2952) I061D10 26-35 50-66 99-114 DHYDILTGLYYYGMDV (SEQ ID NO: 2760) I061E01 26-35 50-66 99-114 LNLEKTVVRGFGYFDL (SEQ ID NO: 2952) I061E05 26-35 50-66 99-115 GGELVWFGESDYYGMDV (SEQ ID NO: 2787) I061E09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I061E12 26-35 50-66 99-106 SQRLFIDS (SEQ ID NO: 2842) I061F01 26-35 50-66 99-119 DRYYDILTGYYIPGLDDAFDI (SEQ ID NO: 2887) I061F09 26-35 50-66 99-112 DSDARLAALDAFDI (SEQ ID NO: 2978) I061F10 26-35 50-66 99-118 EESYYDILTGYYVHYYGMDV (SEQ ID NO: 2743) I061F11 26-35 50-66 99-114 ATYDPLTGYYFDGFDI (SEQ ID NO: 2949) I061G01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I061G03 26-35 50-66 99-114 AYYDILTGFLPYDMDL (SEQ ID NO: 2771) I061G09 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I061G10 26-35 50-65 98-116 EGSYDILTGYYVGVGRMDV (SEQ ID NO: 2171) I061G11 26-35 50-68 101-110  RDILTGFYDS (SEQ ID NO: 2933) I061H05 26-37 52-67 100-115  ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I064A05 26-35 50-68 101-115  DFYDILTGYQHGMDV (SEQ ID NO: 2919) I064A11 26-35 50-66 99-111 HSKEYNWNYALDY (SEQ ID NO: 2754) I064B01 26-35 50-66 99-111 TRMDVLTRYYSDF (SEQ ID NO: 2750) I064B02 26-35 50-66 99-117 AFEDYDILTGYYHHDAFDI (SEQ ID NO: 2911) I064B12 26-35 50-66 99-106 PSYHYMDV (SEQ ID NO: 2740) I064C06 26-35 50-66 99-118 VNADYDILTGYPRDYYGMDV (SEQ ID NO: 2819) I064D01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I064D02 26-35 50-66 99-119 EDATYYDILTGYYMGSYGMDV (SEQ ID NO: 2763) I064E01 26-35 50-66 99-116 ETRKYTSSPPYNYYYMDV (SEQ ID NO: 2736) I064E02 26-35 50-66 99-113 RDYDILTGYSRGFDP (SEQ ID NO: 2725) I064E03 26-35 50-66 99-117 DGIYDILTTLVSYYNGMDV (SEQ ID NO: 2775) I064E07 26-35 50-65 98-113 GERDILTGYYLDGMDV (SEQ ID NO: 2948) I064E08 26-35 50-66 99-113 ERGSYSSGYSGAFDV (SEQ ID NO: 2898) I064F05 26-35 50-66 99-115 ESGGYSYGSRDYYGMDV (SEQ ID NO: 2836) I064F08 26-35 50-66 99-118 DRGVGYDILTGRTYYYGMDV (SEQ ID NO: 2900) I064G06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I065A12 26-35 50-66 99-116 DVSGHDILTGYSYRYFDV (SEQ ID NO: 2795) I065C04 26-35 50-66 99-112 GQKNYYESSGYLEH (SEQ ID NO: 2916) I065C09 26-35 50-66 99-113 GDYDILTGYYSHFDY (SEQ ID NO: 2908) I065E02 26-35 50-66 99-114 AYDYDILTGYSYYFDY (SEQ ID NO: 2895) I065E04 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I065F03 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I065G06 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I065G07 26-35 50-66 99-115 GGNYDILTGYYIGAFDI (SEQ ID NO: 2824) I065G08 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I065H06 26-35 50-66 99-117 GYEYYDILTGYNELGAFDI (SEQ ID NO: 2851) I066A03 26-35 50-66 99-117 DGTYYDILTGYYNQYGMDV (SEQ ID NO: 2915) I066A08 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I066A09 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I066A10 26-35 50-66 99-115 DRGYDILTGYYYYGMDV (SEQ ID NO: 2876) I066A11 26-35 50-66 99-116 EVRDYDILTGYYISYMDV (SEQ ID NO: 2778) I066B02 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I066B08 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I066B10 26-35 50-66 99-115 GLYFEDTNYRHGDAFDI (SEQ ID NO: 2790) I066C02 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I066C11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I066C12 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I066D06 26-35 50-66 99-113 ENYDFLTGYYGAFDI (SEQ ID NO: 2772) I066D08 26-35 50-66 99-111 HSKEYNWNYALDY (SEQ ID NO: 2754) I066D11 26-35 50-66 99-117 ERSQFDFLTGVDRYHPMDV (SEQ ID NO: 2956) I066D12 26-35 50-66 99-112 EGAADYLNGQYFQH (SEQ ID NO: 2815) I066E06 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I066E12 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I066G05 26-35 50-66 99-115 GLYFEDTNYRHGDAFDI (SEQ ID NO: 2790) I066G08 26-35 50-66 99-114 VYYDILTGHPTYGMDV (SEQ ID NO: 2791) I066G10 26-35 50-68 101-117  GIYDILTGYHWDDAFDI (SEQ ID NO: 2872) I066G12 26-35 50-66 99-116 ESTYDILTGSYHDYGLDV (SEQ ID NO: 2822) I066H04 26-35 50-65 98-116 DRLHYDILTGHQTDDAFDI (SEQ ID NO: 2885) I067A07 26-35 50-66 99-117 VLTNYDILTGYYREDAFDM (SEQ ID NO: 2939) I067A11 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I067B08 26-35 50-66 99-122 DRGASNYDILTGYYAPAQGVAFDI (SEQ ID NO: 2969) I067C08 26-37 52-69 102-121  EGAHYDILTGHNYYHYGMDV (SEQ ID NO: 2747) I067C09 26-35 50-66 99-116 ETRKYTSSPPYNYYYMDV (SEQ ID NO: 2736) I067D07 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I067E01 26-35 50-66 99-113 DQHDILTGVYYGMDV (SEQ ID NO: 2921) I067E06 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I067E07 26-35 50-67 100-123  DYPGSEYDILTGYLFGYYYYGMDV (SEQ ID NO: 2926) I067E11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I067G03 26-35 50-66 99-113 ARRVGVLGGKNAFEI (SEQ ID NO: 2765) I067G05 26-35 50-66 99-113 DQHDILTGGYYGMDV (SEQ ID NO: 2894) I067G12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I067H05 26-35 50-68 101-119  EGTYYDILTGYYPLGYFDY (SEQ ID NO: 2936) I067H06 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I068C09 26-35 50-66 99-110 GGSSQNFYGMDV (SEQ ID NO: 2884) I068G03 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I068G04 26-35 50-66 99-115 GVVWVAYGDVGIYGFDV (SEQ ID NO: 2937) I068G07 26-35 50-66 99-113 HDYYIMTAAHYYYDS (SEQ ID NO: 2909) I068G08 26-35 50-66 99-116 GIGYDLLTGYFTGSPLDY (SEQ ID NO: 2846) I070F07 26-35 50-66 99-113 DFYDILTGYHDAFDI (SEQ ID NO: 2910) I070G05 26-35 50-68 101-113  DVDDILTGYSWDY (SEQ ID NO: 2867) I070H02 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I071A01 26-35 50-66 99-114 AAYDPLTGYSFDGFDI (SEQ ID NO: 2783) I071A03 26-35 50-66 99-116 DMHYDILTGYYTGLAFDM (SEQ ID NO: 2917) I071B08 27-36 51-67 100-115  GGYDILTQYPAEFFHP (SEQ ID NO: 2764) I071E01 26-35 50-66 99-111 DFGVIGDYRPFDY (SEQ ID NO: 2777) I071F11 26-35 50-66 99-108 SSNPVYGLDV (SEQ ID NO: 2957) I071G11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I071H08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I074A02 26-35 50-66 99-114 DDRDILTNYYLEYFQH (SEQ ID NO: 2868) I074A08 26-35 50-66 99-120 SSPPKWYDALTGDSSYHSAMDV (SEQ ID NO: 2165) I074D10 26-35 50-66 99-117 DKTLGDQLVEAYYYDGMDV (SEQ ID NO: 2776) I074E01 26-35 50-66 99-117 LGRTSRDLLTGYHFYNMDV (SEQ ID NO: 2944) I074E02 26-35 50-66 99-113 DDYDILTGSLYYFDS (SEQ ID NO: 2803) I074E08 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I074F12 26-35 50-66 99-113 DRADILTGYNDAFDI (SEQ ID NO: 2739) I074H06 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I074H07 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I074H08 26-35 50-66 99-115 VSNDILTGWGGYNWFDP (SEQ ID NO: 2955) I075A07 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I075B01 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075B04 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075B06 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I075B08 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I075B09 26-35 50-66 99-114 TYYDILTGYYAEYFQH (SEQ ID NO: 2932) I075B12 26-35 50-66 99-113 SDYDILTGYYWVPAV (SEQ ID NO: 2812) I075C01 26-35 50-66 99-120 GREDTDKVKPWDRYFHYYYMDV (SEQ ID NO: 2835) I075C05 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075D05 26-35 50-66 99-116 GTGYDILTGYYMGSVFDP (SEQ ID NO: 2897) I075D07 26-35 50-66 99-114 SYYDILTGYYHTPLDY (SEQ ID NO: 2853) I075D08 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I075E01 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I075E03 28-37 52-68 101-121  GGGYDILTGYSYPYLYYGLDV (SEQ ID NO: 2865) I075E04 26-35 50-66 99-116 GRGYDVLTGYFTGSPLDY (SEQ ID NO: 2881) I075E05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I075E10 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I075E11 26-35 50-66 99-106 SGPGWFDP (SEQ ID NO: 2870) I075E12 26-35 50-66 99-115 TDRFGAKDVTARWGMDV (SEQ ID NO: 2979) I075F02 26-35 50-66 99-117 EQGYDILTGYYPEGGWFDP (SEQ ID NO: 2834) I075F04 26-37 52-67 100-114  AGYDLLTGYPFYFDS (SEQ ID NO: 2757) I075F06 26-35 50-66 99-117 GRNYYDFLTGYNFNLGLDY (SEQ ID NO: 2830) I075F07 26-35 50-66 99-113 ENYDSLTGYYNYFDY (SEQ ID NO: 2971) I075F08 26-35 50-66 99-106 DQRKAQDI (SEQ ID NO: 2779) I075F09 26-35 50-66 99-118 LKAPYYDLLTGYHLPKWFDT (SEQ ID NO: 2953) I075F10 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075F11 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075G05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I075G07 26-35 50-66 99-113 GRYYDMLTRGGYFDY (SEQ ID NO: 2858) I075G08 26-35 50-66 99-113 RQYDILTGYYGGFDY (SEQ ID NO: 2958) I075G11 26-35 50-66 99-114 TDYDILTGYPMGYFDP (SEQ ID NO: 2173) I075G12 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075H02 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I075H03 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075H06 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075H08 26-35 50-66 99-116 GSGYDLLTGYFTGSPLDY (SEQ ID NO: 2766) I076A01 26-35 50-66 99-115 DRRRDDLTGYLYDAFDS (SEQ ID NO: 2878) I076A03 26-35 50-68 101-108  GYDTAMQY (SEQ ID NO: 2951) I076A06 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I076A07 26-35 50-66 99-112 DRRDILTGSNFGQD (SEQ ID NO: 2913) I076A08 26-35 50-66 99-115 MGHYDILTGYRHYGMDV (SEQ ID NO: 2831) I076B01 26-35 50-66 99-116 GSGYDLLTGYFTGSPLDY (SEQ ID NO: 2766) I076B03 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I076B07 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I076B08 26-35 50-66 99-114 PYYDPLTAYTFQYFGN (SEQ ID NO: 2806) I076C04 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I076C10 26-35 50-66 99-113 GRYYDMLTRGGYFDY (SEQ ID NO: 2858) I076D01 26-35 50-66 99-114 LDYDILTGYYPSGFDY (SEQ ID NO: 2799) I076D08 26-37 52-67 100-113 RFYDLLTGYSAFDS (SEQ ID NO: 2756) I076D11 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I076D12 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I076E04 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I076E07 26-35 50-66 99-113 EYYDVLTGLFYYMDV (SEQ ID NO: 2841) I076E09 26-35 50-66 99-114 DDRDILTNYYLEYFQH (SEQ ID NO: 2868) I076E11 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I076F01 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I076F03 26-36 51-66 99-113 GDYDVLTGYLRKLDY (SEQ ID NO: 2742) I076F04 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I076F08 26-36 51-66 99-113 VHYDILTGYLWAFDI (SEQ ID NO: 2730) I076F10 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I076G09 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I076G10 26-35 50-66 99-113 GRYYDMLTRGGYFDY (SEQ ID NO: 2858) I076G11 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I076G12 26-35 50-66 99-119 NGYYDILTGYYLWDYYYGMDV (SEQ ID NO: 2769) I076H02 26-35 50-66 99-113 ENYDSLTGYYNYFDY (SEQ ID NO: 2971) I076H04 26-35 50-66 99-114 THYDILTGYYSHPLDY (SEQ ID NO: 2863) I076H05 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I076H06 26-35 50-66 99-113 VPYDILTGYWGAFDV (SEQ ID NO: 2827) I076H09 26-35 50-66 99-116 GSGYDLLTGYFTGSPLDY (SEQ ID NO: 2766) I076H10 26-35 50-66 99-116 GSGYDLLTGYFTGSPLDY (SEQ ID NO: 2766) I077D06 26-35 50-66 99-113 VYYDILTGYNLFFDY (SEQ ID NO: 2177) I078B04 26-35 50-66 99-113 VYYDILTGYNLFFDY (SEQ ID NO: 2177) I078E10 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I002A01-K 26-35 50-66 99-114 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I002A01-R 26-35 50-66 99-114 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I026C04-K 26-35 50-66 99-114 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I026C04-R 26-35 50-66 99-114 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I067B10 26-35 50-66 99-122 DRGAPNYDILTGYYAPAQGVAFDI (SEQ ID NO: 2176) I068C06 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075F12 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I003C06 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I025B06 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I025B09 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I026C04 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I027B12 26-34 49-65 99-114 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I030A10 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I064C04 26-35 50-66 99-120 DGRLSYDILTGYYARDYYGMDD (SEQ ID NO: 2188) I064C07 26-35 50-66 99-107 SEGTIFGVD (SEQ ID NO: 2178) I065D04 26-36 51-66 99-117 GKGYYDILTGYYRDNWFDP (SEQ ID NO: 2181) I065D08 26-35 50-66 99-120 TPSSVYDLLTGYYHYFYSYMDV (SEQ ID NO: 2189) I065F08 26-35 50-66 99-108 EKSAAGYFDY (SEQ ID NO: 2190) I067F05 26-35 50-66 99-113 ENYDSLTGYYGAFDI (SEQ ID NO: 2185) I068B04 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I068B08 26-34 49-65 98-113 KLGLSIVGATTGALDM (SEQ ID NO: 2186) I068C08 26-35 50-66 99-115 EGMNDFINSHHYYTMDA (SEQ ID NO: 2182) I068F03 26-35 50-66 99-112 AGNEYGHTERPADY (SEQ ID NO: 2180) I069B07 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I071B03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072B09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073F04 26-35 50-66 99-109 SLATRPLGMDV (SEQ ID NO: 2184) I074B12 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I075A02 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I075G01 26-35 50-66 99-113 DHFDTLTGYFRRLDS (SEQ ID NO: 2187) I078D02 26-35 50-66 99-113 VYYDILTGYNLFFDY (SEQ ID NO: 2177) I078D08 26-35 50-66 99-117 DAQSYYDILTGYQSYAFDI (SEQ ID NO: 2183) I078H08 26-35 50-66 99-113 VYYDILTGYNLFFDY (SEQ ID NO: 2177) I064A03 26-35 50-66 99-123 GPSTTYYDILTGYYTPYYYYYYMDV (SEQ ID NO: 3014) I064B03 26-37 52-67 100-118  HVRDYDILTGYYRGHYFDY (SEQ ID NO: 2167) I064B05 26-35 50-66 99-113 ERGVVTAYGGDSFDL (SEQ ID NO: 2985) I064B11 26-35 50-66 99-111 DRGPGLLSSFFES (SEQ ID NO: 3033) I064C02 26-35 50-66 99-119 DEYYDILTGYQAPYYYYGMDV (SEQ ID NO: 3068) I064C03 26-35 50-66 99-113 ERGVVTAYGGDSFDL (SEQ ID NO: 2985) I064C11 26-35 50-65 98-116 DVTYHDILTGYAGHEAFDI (SEQ ID NO: 3055) I064C12 26-37 52-69 102-121  ESGRYDILTGYYSGGGGMDV (SEQ ID NO: 3012) I064D03 26-35 50-66 99-119 DGANYDILTGYYTTTVYGMDV (SEQ ID NO: 3072) I064D04 26-35 50-66 99-114 RSYDILTGYYTYGMDV (SEQ ID NO: 3090) I064D06 26-35 50-66 99-107 EGSSGYLVG (SEQ ID NO: 2981) I064E05 26-37 52-67 100-119  KQRGDYDILTGYQLGYAFDI (SEQ ID NO: 2808) I064E06 26-35 50-66 99-118 ERPGYDILTGYPSSIYGMDV (SEQ ID NO: 3053) I064F07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I064F09 26-35 50-66 99-120 DTLGYDILTGYPPPYYYYDMDV (SEQ ID NO: 2988) I064F10 22-31 46-62 95-116 DTLGYDILTGYPPPYYYYDMDV (SEQ ID NO: 2988) I064F11 26-35 50-65 98-115 GRHYYDILTGYYNEAFDI (SEQ ID NO: 3031) I064G01 26-35 50-66 99-113 NYYDVLTQSYYGMDV (SEQ ID NO: 3077) I064G04 26-35 50-66 99-106 DNSGTYGY (SEQ ID NO: 3084) I064G08 26-35 50-66 99-111 GGVTAGRSVYFDS (SEQ ID NO: 2990) I064G10 26-35 50-66 99-113 SPNGDYSGYAWGLEY (SEQ ID NO: 3085) I064G11 26-35 50-65 98-111 YFDGSGYYPVSFSY (SEQ ID NO: 3064) I064G12 26-35 50-65 98-112 VNYDILTGLGYYFDY (SEQ ID NO: 3049) I064H03 26-37 52-67 100-116  SYYDILTGRPYTDAFDI (SEQ ID NO: 2989) I064H04 26-35 50-66 99-115 PLGITAVRGAKTDAFGI (SEQ ID NO: 2929) I064H06 26-35 50-66 99-122 DRGASNYDILTGYYAPAQGVAFDI (SEQ ID NO: 2969) I065A02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I065A04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I065A06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I065A07 26-35 50-66 99-117 DGGGYDILTGYQYYYGMDV (SEQ ID NO: 2987) I065B01 26-35 50-65 98-118 WATYYDTLTGYRLKDHAGFDI (SEQ ID NO: 3017) I065B05 26-35 50-66 99-115 SPGDDILTGYYKYYFDY (SEQ ID NO: 3032) I065B09 26-35 50-66 99-119 DAGESYDILTGYYVIEGYMDV (SEQ ID NO: 2986) I065B12 26-35 50-66 99-112 EGAADYLNGQYFQH (SEQ ID NO: 2815) I065C02 26-35 50-66 99-109 EGSWSGLDLDY (SEQ ID NO: 3007) I065C06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I065C08 26-35 50-66 99-114 VSGYNSGYFESYDMDV (SEQ ID NO: 2732) I065C10 26-35 50-66 99-110 QGGQYDSPPLDV (SEQ ID NO: 3002) I065D01 26-35 50-66 99-115 DRDYDILTDYSNYGMDV (SEQ ID NO: 3074) I065D03 26-35 50-66 99-115 APLYDILTGYYIGGNDY (SEQ ID NO: 3028) I065D05 26-35 50-66 99-116 DKDYDILTGYWRDELLDY (SEQ ID NO: 3040) I065D06 26-35 50-66 99-115 DPNYDILTGYYYYAMDV (SEQ ID NO: 3062) I065E01 26-35 50-66 99-112 EFDQLLARGHGMDV (SEQ ID NO: 3027) I065E05 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I065E06 26-35 50-66 99-119 ARGSYDILTGYYRPGDGYFDY (SEQ ID NO: 3043) I065E08 26-35 50-66 99-115 GLYFEDTNYRHGDAFDI (SEQ ID NO: 2790) I065E09 26-35 50-65 98-118 ERSYYDILTGYSPRSKYGMDV (SEQ ID NO: 3021) I065E12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I065F04 26-35 50-66 99-113 ERGVVTAYGGDSFDL (SEQ ID NO: 2985) I065F05 26-35 50-65 98-113 RYSDALTGYSLGAFDV (SEQ ID NO: 3018) I065F07 26-38 53-69 102-118 GAYYDILTGYYPYGMDV (SEQ ID NO: 2860) I065F09 26-35 50-66 99-116 DYPIDVLTGRRTKNWFDP (SEQ ID NO: 3013) I065F12 26-35 50-66 99-114 DQVDRLLMQYNYYMDA (SEQ ID NO: 3047) I065G01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I065G09 26-35 50-68 101-116  DAYYDILTGWVYGMDV (SEQ ID NO: 3030) I065G10 26-36 51-66 99-113 FRYDILTGYYYDMDV (SEQ ID NO: 2983) I065H05 26-35 50-66 99-113 EYYDILTGYSGAFDI (SEQ ID NO: 2984) I065H07 26-35 50-66 99-111 TRMDVLTRYYSDF (SEQ ID NO: 2750) I066A05 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I066A06 26-35 50-66 99-112 EGAADYLNGQYFQH (SEQ ID NO: 2815) I066A12 26-35 50-66 99-115 DTRVIGIQLWERGAFDM (SEQ ID NO: 3080) I066B05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I066B11 26-35 50-66 99-115 PLGITAVRGAKTDAFGI (SEQ ID NO: 2929) I066C06 26-35 50-65 98-117 GRRYYDILTGYSLGRGEMDV (SEQ ID NO: 3009) I066C10 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I066D02 26-35 50-66 99-110 AGTSLMNYGTDV (SEQ ID NO: 3048) I066D07 26-35 50-66 99-114 GPYDVLTGYLSGNFDY (SEQ ID NO: 2992) I066E01 26-35 50-66 99-110 QGGQYDSPPFDV (SEQ ID NO: 3001) I066E03 26-35 50-66 99-122 GEKARYYDILTGYYSAWGGYYMDV (SEQ ID NO: 3045) I066E04 26-35 50-66 99-114 LNLEKTVIRGFGYFDL (SEQ ID NO: 3081) I066E05 26-35 50-66 99-115 VGGYDILTGYYLRGMDV (SEQ ID NO: 2997) I066E07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I066E09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I066F01 26-35 50-66 99-114 SPYDTLTGYVYNGVDV (SEQ ID NO: 3058) I066F03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I066F04 26-35 50-66 99-114 VAAAGARTLGYFGMDV (SEQ ID NO: 3071) I066F07 26-35 50-66 99-116 DVSGHDILTGYSYRYFDV (SEQ ID NO: 2795) I066F08 26-35 50-66 99-117 SPMYYDRLTGFYPSGYFDS (SEQ ID NO: 3036) I066F11 26-35 50-66 99-115 GAYYDILTGYYPYGMDV (SEQ ID NO: 2860) I066F12 26-35 50-66 99-114 GPSSAGTTIGLGSFDP (SEQ ID NO: 3005) I066G06 26-35 50-66 99-116 ETRKYTSSPPYNYYYMDV (SEQ ID NO: 2736) I066G07 26-30 45-61 94-106 DQFSVGGRHAFDL (SEQ ID NO: 3054) I066H02 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I067A02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I067A03 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I067A06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I067A08 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I067A10 26-35 50-66 99-113 ERGVVTAYGGDSFDL (SEQ ID NO: 2985) I067B03 26-35 50-66 99-115 PLGITAVRGAKTDAFGI (SEQ ID NO: 2929) I067B04 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I067C03 26-35 50-66 99-106 DWGHWFDP (SEQ ID NO: 2982) I067C05 26-35 50-66 99-110 SGSSLMTYGTDV (SEQ ID NO: 3015) I067C07 26-35 50-66 99-114 EPYDILTGYYGSYFDY (SEQ ID NO: 3041) I067C10 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I067C12 26-35 50-66 99-115 TYYDILTGYSGGGAFDY (SEQ ID NO: 3024) I067D01 26-35 50-66 99-109 GSRVRGVTPDL (SEQ ID NO: 3020) I067D03 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I067D05 26-35 50-66 99-119 ECSGSSCPARQPPYYQYYMDV (SEQ ID NO: 2993) I067D06 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I067D09 26-35 50-66 99-115 GAYYDILTGYYPYGMDV (SEQ ID NO: 2860) I067D12 26-35 50-66 99-110 QGGQYDSPPLDV (SEQ ID NO: 3002) I067E02 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I067E04 26-35 50-66 99-115 GAYYDILTGYYPYGMDV (SEQ ID NO: 2860) I067E05 26-35 50-66 99-117 DYRNYDILTGHPYYYGMDV (SEQ ID NO: 2996) I067F01 26-35 50-66 99-114 QHYDILTGYSQEPFDI (SEQ ID NO: 3022) I067F03 26-35 50-66 99-117 DQTYYDILTGHYYYYGMDV (SEQ ID NO: 3087) I067F04 26-35 50-66 99-112 EGAADYLNGQYFQH (SEQ ID NO: 2815) I067F08 26-35 50-66 99-113 LGYYDILTGYRSDDY (SEQ ID NO: 3029) I067F10 26-35 50-66 99-110 AGSSLMAYGTDV (SEQ ID NO: 3016) I067F11 26-35 50-66 99-113 ENYDFLTGYYGAFDI (SEQ ID NO: 2772) I067G01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I067G09 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I067H07 26-35 50-66 99-117 GGLYDILTGRPATDDAFDI (SEQ ID NO: 3035) I068A07 26-35 50-66 99-115 TDRFGAKDVTARWGMDV (SEQ ID NO: 2979) I068E05 26-35 50-66 99-120 GREDTDKVKPWDRYYHYYYMDV (SEQ ID NO: 2809) I068E08 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I068E11 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I068F04 26-35 50-66 99-114 ELGHREGGYWYSPYNV (SEQ ID NO: 2838) I068G05 26-35 50-66 98-108 KNMGASAAADF (SEQ ID NO: 3042) I068G06 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I068G11 26-35 50-66 99-119 ESGSHYDLLTGLLVAANGFDV (SEQ ID NO: 3044) I069A09 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I069A10 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I069B06 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I069B09 26-35 50-66 99-112 PYYDILTGYFAFDI (SEQ ID NO: 3026) I069B12 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I069C06 26-35 50-66 99-116 VLPHYDILTGYSQNWFDP (SEQ ID NO: 3000) I069C09 26-35 50-66 99-116 VLPHYDILTGYSQNWFDP (SEQ ID NO: 3000) I069D03 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I069E09 26-35 50-66 99-115 DGYYDILTGYSYYGMDV (SEQ ID NO: 2135) I069E11 26-35 50-66 99-113 VYYDILTGYNLFFDY (SEQ ID NO: 2177) I069F05 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I069F07 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I069F12 26-35 50-66 99-113 GYYDILTGYYDAFDI (SEQ ID NO: 3051) I069G06 26-35 50-66 99-115 DGYYDILTGYSGYYMDV (SEQ ID NO: 3059) I069G08 26-35 50-66 99-118 DRLEYYDILTGYYYYYGMDV (SEQ ID NO: 3039) I069G11 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I070A03 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I070A09 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I070B01 26-35 50-66 99-117 SQSDYDILTGYYYYYGMDV (SEQ ID NO: 3038) I070B05 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I070D03 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I070D04 26-35 50-66 99-114 MEYDILTSYYGGYFDY (SEQ ID NO: 3034) I070E01 26-35 50-66 99-117 SQSDYDILTGYYYYYGMDV (SEQ ID NO: 3038) I070F01 26-35 50-66 99-117 SQSNYDILTGYYYYYGMDV (SEQ ID NO: 3067) I070G10 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I071A06 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I071B02 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I071D02 26-35 50-66 99-110 AGTSLMNYGTDV (SEQ ID NO: 3048) I071D08 26-37 52-66 99-119 VPYYYDTSGGYLGEYYYGMDV (SEQ ID NO: 3010) I071F01 26-35 50-66 99-110 AGTSLMNYGTDV (SEQ ID NO: 3048) I071G09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072A01 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I072A09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072B02 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I072B10 26-35 50-66 99-110 AGSSLMTYGTDV (SEQ ID NO: 2773) I072B11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072B12 26-35 50-66 99-113 ENYDYLTGYYGAFDI (SEQ ID NO: 2995) I072C05 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I072C10 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072D01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072D05 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I072E01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072E04 26-35 50-66 99-117 EGSYDILTGYYVGVGRMDV (SEQ ID NO: 2171) I072E05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072E06 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I072F03 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I072F07 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072F11 26-35 50-66 99-113 DEYDILTGLLQGMDV (SEQ ID NO: 2883) I072G03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072G04 26-35 50-68 101-110  RDILTGFYDS (SEQ ID NO: 2933) I072G05 26-35 50-66 99-110 GYRNDWYGAFEI (SEQ ID NO: 3079) I072G09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072H03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I072H07 26-35 50-66 99-110 AGTSLMNYGMDV (SEQ ID NO: 3070) I073A02 26-35 50-66 99-114 GPYDILTGYYRDAFDI (SEQ ID NO: 2998) I073A03 26-35 50-66 99-115 THYDILTGYYTADAFDI (SEQ ID NO: 3019) I073A04 26-35 50-66 99-121 VQMDSEYYDLLTGINVGPYYFDY (SEQ ID NO: 2132) I073A05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073A06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073A09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073A10 26-35 50-66 99-119 GDFGDYDILTGYYPVYYGMDV (SEQ ID NO: 3082) I073A11 26-35 50-66 99-114 SYYDILTGYYPFGMDV (SEQ ID NO: 3004) I073B02 26-35 50-66 99-117 DLWYYDILTGYYLDDAFDI (SEQ ID NO: 2999) I073B05 26-35 50-66 99-117 DLWYYDILTGYYLDDAFDI (SEQ ID NO: 2999) I073B06 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I073B07 26-35 50-66 99-111 TRMDVLTRYYSDF (SEQ ID NO: 2750) I073B08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073B11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073C01 26-35 50-66 99-114 GYHDTLTSYNYNWFDP (SEQ ID NO: 3006) I073C02 26-35 50-66 99-121 AQMDSEYYDLLTGINVGPYYFDY (SEQ ID NO: 3076) I073C04 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073C07 26-35 50-66 99-107 GMGDHYMDV (SEQ ID NO: 3008) I073C08 26-35 50-66 99-115 EMGYDILTGYYLNYMDV (SEQ ID NO: 2862) I073C09 26-35 50-66 99-114 QHYDILTGYSQEPFDI (SEQ ID NO: 3022) I073C11 26-35 50-68 101-119  FNPTYDILTGYYIGGYFQH (SEQ ID NO: 2155) I073C12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073D01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073D03 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I073D06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073D08 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I073D10 26-35 50-68 101-113  QYYDILTGYELDI (SEQ ID NO: 3073) I073D11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073E01 26-37 52-69 102-121  EGAHYDILTGHNYYHYGMDV (SEQ ID NO: 2747) I073E02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073E03 26-35 50-66 99-114 ATYDPLTGYSLDGFDI (SEQ ID NO: 3003) I073E05 26-35 50-66 99-114 QHYDILTGYSQEPFDI (SEQ ID NO: 3022) I073E06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073E08 26-35 50-66 99-113 ENYDFLTGYYGAFDI (SEQ ID NO: 2772) I073F01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073F02 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073F03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073F05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073F07 26-35 50-66 99-114 GEYDILTGYPYWYFDL (SEQ ID NO: 3023) I073F09 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073F11 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073F12 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073G03 26-35 50-66 99-116 DGSYDILTGYYIDNYMDV (SEQ ID NO: 2154) I073G04 26-35 50-65 98-116 GEGGYDILTGYLRGYGMDV (SEQ ID NO: 3037) I073G05 26-35 50-66 99-108 GMGDHYGMDV (SEQ ID NO: 2161) I073G06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073G07 26-35 50-66 99-115 GSYYDILTGISSLGMDV (SEQ ID NO: 3063) I073G08 26-35 50-66 99-112 SRDLLLFPHYGMDV (SEQ ID NO: 2133) I073G09 26-35 50-66 99-118 DRGHYDILTGYYIEPSGFDY (SEQ ID NO: 3061) I073G10 26-35 50-66 99-108 GPGVIGNYDY (SEQ ID NO: 2749) I073G12 26-35 50-68 101-115  GGMIRAREDYYYMDV (SEQ ID NO: 3083) I073H01 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073H03 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073H05 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073H06 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I073H07 26-35 50-66 99-111 TYYDILTGYYFDY (SEQ ID NO: 3056) I073H08 26-35 50-66 99-114 ATYDPLTGYSFDGFDI (SEQ ID NO: 2153) I074A05 26-35 50-66 99-116 LPPYDMLTGYYVGGGMDV (SEQ ID NO: 3050) I074A06 26-35 50-66 99-116 AKPYTDFSRGSDADAFDV (SEQ ID NO: 3065) I074B03 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I074B11 26-35 50-66 99-112 RYGDPFYYYYYMNV (SEQ ID NO: 2755) I074C07 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I074D03 26-35 50-66 99-114 GGYDILTQYPAEFFHP (SEQ ID NO: 2764) I074D04 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I074D05 26-35 50-66 99-116 DRYYDILTKGDYYYGMDV (SEQ ID NO: 3060) I074D07 26-35 50-66 99-123 VQGETYYDILTGYWGPKRDLYGMDV (SEQ ID NO: 3069) I074D08 26-34 49-65 98-113 ELGLSIVVATTGALDM (SEQ ID NO: 2980) I074D11 26-35 50-66 99-111 ESEGGDYTNPFGY (SEQ ID NO: 2991) I074E05 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I074E07 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I074E09 26-35 50-68 101-119  DPGNYDILTGYYYYYGMDV (SEQ ID NO: 2935) I074E11 26-35 50-66 99-110 VRLPHHHYFMAV (SEQ ID NO: 3075) I074H05 26-35 50-66 99-115 ESSITVNPPYYFYGMDV (SEQ ID NO: 3025) I075A03 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075A10 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I075B07 26-35 50-66 99-116 SPEGDYQPLSSNYNWLDP (SEQ ID NO: 3011) I075D11 26-36 51-66 99-106 GKEGYNDN (SEQ ID NO: 3089) I075D12 26-35 50-66 99-116 GSGYDLLTGYFTGSPLDY (SEQ ID NO: 2766) I075G02 26-35 50-66 99-116 SPEGDYQPLSSNYNWLDP (SEQ ID NO: 3011) I075G09 26-35 50-66 99-115 MGHYDILTGYRHYGMDV (SEQ ID NO: 2831) I075G10 26-35 50-66 99-111 GNYDILTGYPHDL (SEQ ID NO: 3086) I075H05 26-35 50-66 99-114 SYYDILTGYYHTPLDY (SEQ ID NO: 2853) I075H07 26-35 50-66 99-116 GSGYDLLTGYFTGSPLDY (SEQ ID NO: 2766) I076A11 26-35 50-66 99-114 DDRDILTNYYLEYFQH (SEQ ID NO: 2868) I076A12 26-35 50-66 99-116 GSGYDVLTGYFTGSPLDY (SEQ ID NO: 3057) I076B06 26-35 50-66 99-113 GRYDILTGYFTSFDY (SEQ ID NO: 3066) I076B10 26-35 50-66 99-114 DDRDILTNYYLEYFQH (SEQ ID NO: 2868) I076B12 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I076C06 26-35 50-66 99-115 MGHYDILTGYRHYGMDV (SEQ ID NO: 2831) I076C11 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I076D06 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I076E05 26-35 50-66 99-116 GTGYDILTGYYMGSAFDQ (SEQ ID NO: 2800) I076E08 26-35 50-66 99-106 DQGRYLDL (SEQ ID NO: 2175) I076F06 26-36 51-66 99-106 RDVQGAPY (SEQ ID NO: 3088) I076G01 26-35 50-66 99-116 VEGVYDILTGYSFDAFDI (SEQ ID NO: 3078) I076H01 26-35 50-66 99-117 EQGYDILTGYYPEGGWFDP (SEQ ID NO: 2834) I076H03 26-34 49-65 98-113 ELGLSIVGATTGALDM (SEQ ID NO: 2174) I077B05 26-37 52-69 102-120  DKSYYDILTGYYYYYGMDV (SEQ ID NO: 3052) I077C10 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I077D01 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I077D04 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I077D11 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I077D12 26-35 50-66 99-113 EKYDILTGYYDAFDI (SEQ ID NO: 3046) I077E01 26-35 50-66 99-115 EMGYDILTGYYLNYMDV (SEQ ID NO: 2862) I077E03 26-35 50-66 99-115 EMGYDILTGYYLNYMDV (SEQ ID NO: 2862) I077E08 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I077F05 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I077G06 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I077H02 26-35 50-66 99-114 MEYDILTGYYGGYFDY (SEQ ID NO: 2179) I078B05 26-35 50-66 99-116 ESHYDILTGYYSNPSFDI (SEQ ID NO: 2994) I079E02 26-35 50-66 99-110 DSGSYYYDAFDI (SEQ ID NO: 2194) I079F11 26-35 50-66 99-105 TGSGFDY (SEQ ID NO: 2192) I082G02 26-35 50-66 99-109 DGYRTNDALDI (SEQ ID NO: 2191) I082H08 26-35 50-66 99-104 DWDMDV (SEQ ID NO: 2193) I099D03 26-35 50-66 99-109 DNGGGTIGFDY (SEQ ID NO: 2195) I079B05 26-35 50-66 99-103 FVLDY (SEQ ID NO: 2210) I079B12 26-35 50-66 99-107 WTSSGAFDI (SEQ ID NO: 2205) I079C01 26-35 50-66 99-104 DWDMDV (SEQ ID NO: 2193) I079F06 26-35 50-66 99-107 DNLHAAFDI (SEQ ID NO: 2202) I079F08 26-35 50-66 99-111 YYYHSSGSDAFDI (SEQ ID NO: 2206) I080A03 26-35 50-66 99-111 VGIKAAAVDNFEY (SEQ ID NO: 2197) I080A08 26-35 50-66 99-108 VHSTGYAFEN (SEQ ID NO: 2200) I080B01 26-35 50-66 99-115 EYSGYHYVEGGSYAMDV (SEQ ID NO: 2201) I080D03 26-35 50-66 99-111 VGIKAAAVDNFEY (SEQ ID NO: 2197) I080E05 26-35 50-66 99-114 EGGGDAYDVAPYYFDY (SEQ ID NO: 2204) I080G07 26-35 50-66 99-109 EGPGYYYGMDV (SEQ ID NO: 2209) I080G09 26-35 50-66 99-109 DNGGGTIGFDY (SEQ ID NO: 2195) I082A05 26-35 50-66 99-104 DLDFDY (SEQ ID NO: 2208) I082B08 26-35 50-66 99-110 DLGIAGTIYFDY (SEQ ID NO: 2207) I082C03 26-35 50-66 99-111 DASRDIVVLPLAI (SEQ ID NO: 2198) I082D07 26-35 50-66 99-107 WTSSGAFDI (SEQ ID NO: 2205) I082G01 26-35 50-66 99-111 DRGSGWPNWYFDL (SEQ ID NO: 2212) I083B12 26-35 50-66 99-110 ESGAGGYYYDDY (SEQ ID NO: 2196) I083G03 26-35 50-66 99-111 VGIKAAAVDNFEY (SEQ ID NO: 2197) I084A01 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084B02 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084C04 25-34 49-65 98-104 NLWGLDY (SEQ ID NO: 2199) I084C11 26-35 50-66 99-107 GNAWGAFDI (SEQ ID NO: 2211) I079A01 26-35 50-66 99-107 EGVAAGEDY (SEQ ID NO: 3123) I079A03 26-35 50-66 99-107 GGMDWDFDY (SEQ ID NO: 3183) I079A04 26-35 50-66 99-107 VDSSGYAYY (SEQ ID NO: 3213) I079A06 26-35 50-66 99-106 DAAVTAEG (SEQ ID NO: 3142) I079A07 26-35 50-66 99-109 GSNYSPDAFDI (SEQ ID NO: 3112) I079A10 26-35 50-68 101-121  LPPDLRYCDGGICPGFDWLGP (SEQ ID NO: 3163) I079A11 26-35 50-66 99-108 GPSYYYYMAV (SEQ ID NO: 3114) I079B02 26-35 50-66 99-107 EGVAAGEDY (SEQ ID NO: 3123) I079B03 26-35 50-66 99-109 GSNYSPDAFDI (SEQ ID NO: 3112) I079B04 26-35 50-66 99-103 LLSDY (SEQ ID NO: 3168) I079B07 26-35 50-66 99-111 DLSGSYFSRYFDY (SEQ ID NO: 3193) I079B09 26-35 50-66 99-112 VEWEDIVVGSAFDI (SEQ ID NO: 3128) I079C02 26-35 50-66 99-117 VTSLYSSSSGGYYYYGMDV (SEQ ID NO: 3145) I079C04 26-35 50-66 99-105 GWRGVDY (SEQ ID NO: 3195) I079C05 26-35 50-66 99-113 AGGNPRSGSLVYFDY (SEQ ID NO: 3225) I079C07 26-35 50-66 99-110 GLDVYAIYGLDV (SEQ ID NO: 3176) I079D01 26-35 50-66 99-117 EVRNYDLLTRSYLAGPLDN (SEQ ID NO: 2751) I079D02 26-35 50-66 99-108 EIGWEGAFDI (SEQ ID NO: 3178) I079D04 26-35 50-66 99-106 VRPGLMDV (SEQ ID NO: 3132) I079D06 26-35 50-66 99-110 EAYTSSWAEFDF (SEQ ID NO: 3190) I079D07 26-35 50-66 99-109 NITPLAMVGDF (SEQ ID NO: 3146) I079D08 26-35 50-66 99-103 LIEDF (SEQ ID NO: 3161) I079D09 26-35 50-66 99-104 DSGSPD (SEQ ID NO: 3108) I079D11 26-35 50-66 99-107 EGVAAGEDY (SEQ ID NO: 3123) I079E06 26-35 50-66 99-109 EKRGSRRVFDI (SEQ ID NO: 3093) I079E08 26-35 50-66 99-110 EAYASSWAEFDF (SEQ ID NO: 3189) I079E11 26-35 50-66 99-109 PYGSGSYAFDI (SEQ ID NO: 3185) I079E12 26-35 50-66 99-116 ARDYYDSSGYYVPDAFDI (SEQ ID NO: 3107) I079F01 26-35 50-66 99-106 GHFYGMDV (SEQ ID NO: 3098) I079F02 26-35 50-68 101-121  LPPDLRYCDGGMCSGFDWLGP (SEQ ID NO: 3219) I079F03 26-35 50-66 99-113 ESLLTEEYCGSDCYS (SEQ ID NO: 3115) I079F04 26-35 50-66 99-109 NSAPPAPSMDV (SEQ ID NO: 3099) I079F09 26-35 50-66 99-103 RYYDY (SEQ ID NO: 3139) I079F10 26-35 50-66 99-109 NITPLAMVGDF (SEQ ID NO: 3146) I079F12 26-35 50-66 99-109 ADYSNDYYMDV (SEQ ID NO: 3166) I079G02 26-35 50-66 99-109 NITPLAMVGDF (SEQ ID NO: 3146) I079G05 26-35 50-66 99-109 FPLESYYYMDV (SEQ ID NO: 3124) I079G06 26-35 50-66 99-108 GNSFGRTLDY (SEQ ID NO: 3158) I079H05 26-35 50-66 99-109 DVPPPDGYLEV (SEQ ID NO: 3192) I079H06 26-35 50-66 99-107 ASYPVPFDY (SEQ ID NO: 3171) I080A01 26-35 50-66 99-104 GGWLDD (SEQ ID NO: 3210) I080A02 26-35 50-66 99-106 EHSSSFDY (SEQ ID NO: 3111) I080A05 26-35 50-66 99-114 EGEGDGYNVAPYYFDY (SEQ ID NO: 3160) I080A06 26-35 50-66 99-114 EAGGSGSYHFSFPFDY (SEQ ID NO: 3188) I080A07 26-35 50-66 99-108 TGIWGYYFDY (SEQ ID NO: 3175) I080A10 26-35 50-66 99-114 DGNLNYDGSTDYGMDV (SEQ ID NO: 3140) I080B02 26-35 50-66 99-111 LGRNYTSSWSLDY (SEQ ID NO: 3181) I080B03 26-35 50-66 99-111 VVGGYSSTLGTDV (SEQ ID NO: 3096) I080B05 26-35 50-66 99-110 LGVARGREAFDL (SEQ ID NO: 3206) I080B06 26-37 52-69 102-115  AVRSPGYYYYYMDV (SEQ ID NO: 3125) I080B07 26-35 50-66 99-106 GRKPLFDY (SEQ ID NO: 3141) I080B08 26-37 52-67 100-109  KQRREKYFDY (SEQ ID NO: 3100) I080B09 26-35 50-66 99-115 EKAIIETTSGEADPFDI (SEQ ID NO: 3151) I080B10 26-37 52-67 100-111  RPALRSLWYFDL (SEQ ID NO: 3102) I080B11 26-35 50-68 101-110  LHCTGGSCGF (SEQ ID NO: 3186) I080B12 26-35 50-66 99-112 NPYYYDSSEGFFDY (SEQ ID NO: 3109) I0800O3 26-35 50-66 99-111 SGRQAYYYYGMDV (SEQ ID NO: 3091) I080006 26-36 51-66 99-117 DYYDGSSYSSGDYYYYMDV (SEQ ID NO: 3227) I080007 26-35 50-66 99-117 DSDLVVIPTAIQGRYYFDN (SEQ ID NO: 3113) I080008 26-35 50-66 99-110 GKRYSYGWYFDI (SEQ ID NO: 3130) I080C10 26-35 50-66 99-104 DTPLDP (SEQ ID NO: 3094) I080C11 26-35 50-66 99-110 EGDPTDNDAFDV (SEQ ID NO: 3155) I080C12 26-35 50-66 99-111 DGPTYARPYYLDH (SEQ ID NO: 3153) I080D01 26-35 50-66 99-109 DGTKYDWGFDY (SEQ ID NO: 3220) I080D02 26-35 50-66 99-114 ETFSHCSGGSCYPFDY (SEQ ID NO: 3212) I080D04 26-35 50-66 99-111 SGRQAYYYYGMDV (SEQ ID NO: 3091) I080D05 26-35 50-66 99-109 EFFGYVYLTDY (SEQ ID NO: 3165) I080D08 26-35 50-68 101-110  LHCTGGSCGF (SEQ ID NO: 3186) I080D09 26-35 50-66 99-111 VDYTDYEMGAFEI (SEQ ID NO: 3187) I080D11 26-35 50-66 99-108 VGNFGYYFEY (SEQ ID NO: 3196) I080D12 26-35 50-68 101-108  SSRNGGDY (SEQ ID NO: 3214) I080E01 26-35 50-66 99-109 DLSRVAGRFDY (SEQ ID NO: 3164) I080E04 26-37 52-67 100-109  HDVYGDLFDY (SEQ ID NO: 3211) I080E06 26-35 50-68 101-110  LHCSGGSCGF (SEQ ID NO: 3221) I080E07 26-35 50-66 99-115 EGSIVGATLTINDAFDI (SEQ ID NO: 3150) I080E08 26-35 50-66 99-110 GKRYSYGWYFDI (SEQ ID NO: 3130) I080E12 26-35 50-66 99-103 DPFDY (SEQ ID NO: 3134) I080F04 26-35 50-66 99-111 DGPTYARPYYLDH (SEQ ID NO: 3153) I080F05 26-35 50-66 99-115 ESSGTLGEFSLELPFDY (SEQ ID NO: 3203) I080F06 26-35 50-66 99-111 LGRNYTSSWSLDY (SEQ ID NO: 3181) I080F08 26-35 50-66 99-103 NAFDY (SEQ ID NO: 3121) I080G03 26-36 51-66 99-113 GRGYSSSSSVYGMDI (SEQ ID NO: 3095) I080G04 26-35 50-66 99-104 VHSSGS (SEQ ID NO: 3216) I080G10 26-35 50-66 99-116 KRGDFGVIRLHHYYGMDV (SEQ ID NO: 3136) I080G11 26-37 52-67 100-109  HDVYGDLFDS (SEQ ID NO: 3205) I080H01 26-37 52-67 100-113  LRPDADYGDYGFDY (SEQ ID NO: 3218) I080H02 26-35 50-66 99-112 TSERGTYRQWDFDN (SEQ ID NO: 3204) I080H03 26-35 50-66 99-108 EAGEVAAIDY (SEQ ID NO: 3180) I080H04 26-35 50-66 99-110 GKRYSYGWYFDI (SEQ ID NO: 3130) I080H05 26-37 52-67 100-109  HDVYGDLFDS (SEQ ID NO: 3205) I080H06 26-35 50-66 99-110 GKRYSYGWYFDV (SEQ ID NO: 3217) I080H07 26-35 50-68 101-110  LHCTGGSCGF (SEQ ID NO: 3186) I080H08 26-35 50-66 99-111 ERGGRDGDYALDF (SEQ ID NO: 3148) I080H09 26-36 51-66 99-112 RTPDHNGDSGPPDY (SEQ ID NO: 3215) I081A01 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081A03 26-35 50-66 99-108 ESLTGGAFDI (SEQ ID NO: 3117) I081A04 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081A06 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081A08 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081A09 26-35 50-66 99-107 GAGSRYFDL (SEQ ID NO: 3118) I081A10 26-35 50-66 99-106 GGDRAFDI (SEQ ID NO: 3119) I081B01 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081B04 26-35 50-66 99-107 GNAWGAFDI (SEQ ID NO: 2211) I081B05 26-35 50-66 99-106 GGDRAFDI (SEQ ID NO: 3119) I081B06 26-35 50-66 99-106 VKRYYFDY (SEQ ID NO: 3179) I081B07 26-35 50-66 99-109 ELTGANDAFDI (SEQ ID NO: 3104) I081B08 26-35 50-66 99-105 RRYALDY (SEQ ID NO: 2920) I081B09 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081B10 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081B11 26-35 50-66 99-105 GFALYKD (SEQ ID NO: 3169) I081C07 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081C08 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081D04 26-35 50-66 99-108 EDLTGDAFDI (SEQ ID NO: 3103) I081D06 26-35 50-66 99-105 GDAYFDY (SEQ ID NO: 3147) I081D08 26-35 50-66 99-105 GDAYFDY (SEQ ID NO: 3147) I081D09 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081D10 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081D11 26-35 50-66 99-107 EGLLDAFDI (SEQ ID NO: 3200) I081D12 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081E02 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081E03 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081E05 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081E06 26-35 50-66 99-107 VGYGGKGDY (SEQ ID NO: 3137) I081E07 26-35 50-66 99-107 GAGSRYFDL (SEQ ID NO: 3118) I081E10 26-35 50-66 99-115 GLAPIVDGGMTNDAFDI (SEQ ID NO: 3184) I081F01 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081F04 26-35 50-66 99-105 RLIRKAR (SEQ ID NO: 3170) I081F05 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081F06 26-35 50-66 99-107 ERGNQAFDI (SEQ ID NO: 3156) I081F07 26-35 50-66 99-105 RRYALDY (SEQ ID NO: 2920) I081F11 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081G01 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081G04 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081G06 26-35 50-66 99-108 SRSPYDAFDI (SEQ ID NO: 3097) I081G10 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081H02 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081H03 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I081H04 26-35 50-66 99-108 SNWGGDAFDI (SEQ ID NO: 3202) I081H06 26-35 50-66 99-103 LAFDI (SEQ ID NO: 3174) I081H08 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I082A02 26-35 50-66 99-112 PAASSRGPKDAFDI (SEQ ID NO: 3129) I082A04 26-35 50-66 99-103 LSGDS (SEQ ID NO: 3122) I082A08 26-35 50-66 99-107 EGVAAGEDY (SEQ ID NO: 3123) I082A11 26-35 50-66 99-103 FVLDY (SEQ ID NO: 2210) I082B06 26-35 50-66 99-104 GNGKDV (SEQ ID NO: 3135) I082B09 26-35 50-66 99-107 EGVAAGEDY (SEQ ID NO: 3123) I082B12 26-35 50-66 99-104 DLDFDY (SEQ ID NO: 2208) I082C01 26-35 50-66 99-109 VNDIVVVDMDV (SEQ ID NO: 3143) I082C05 26-35 50-66 99-109 EKRGSRRVFDI (SEQ ID NO: 3093) I082C08 26-35 50-66 99-110 LSNRNDNLRLDY (SEQ ID NO: 3106) I082D02 26-35 50-66 99-103 FVLDY (SEQ ID NO: 2210) I082E05 26-35 50-66 99-107 TWATNTFDM (SEQ ID NO: 3152) I082E06 26-35 50-66 99-103 FDLDY (SEQ ID NO: 3167) I082E07 26-35 50-66 99-112 VEWEDIVVGSAFDI (SEQ ID NO: 3128) I082F11 26-35 50-66 99-109 GGDMTTVTTDY (SEQ ID NO: 3177) I082G07 26-35 50-66 99-109 ADYSNDYYMDV (SEQ ID NO: 3166) I082G10 26-35 50-66 99-107 EGVAAGEDY (SEQ ID NO: 3123) I082G11 26-35 50-66 99-116 GPIYYFDGSAYEGYYFDY (SEQ ID NO: 3222) I082H04 26-35 50-65 98-105 MNADAFEI (SEQ ID NO: 3223) I082H09 26-35 50-66 99-112 PAASSRGPKDAFDI (SEQ ID NO: 3129) I083A06 26-35 50-66 99-109 DSRPTNRAFHY (SEQ ID NO: 3110) I083A09 26-35 50-68 101-110  LHCTGGSCGF (SEQ ID NO: 3186) I083A11 26-35 50-66 99-108 VRDDSAGFDY (SEQ ID NO: 3173) I083B03 26-35 50-66 99-110 VLVRGQYRGMDL (SEQ ID NO: 3138) I083B05 26-35 50-66 99-111 VDYTDYEMGAFDL (SEQ ID NO: 3172) I083B06 26-35 50-66 99-111 DRIAAAGGDAFDI (SEQ ID NO: 3194) I083B10 26-35 50-66 99-110 DLYKNGYALFDS (SEQ ID NO: 3197) I083C01 26-35 50-66 99-108 DEYSSLYMDV (SEQ ID NO: 3201) I083C02 26-35 50-66 99-108 FGAGRLYDDY (SEQ ID NO: 3224) I083C07 26-35 50-66 99-109 DNGGGTIGFDY (SEQ ID NO: 2195) I083C12 26-35 50-66 99-108 DQGIETANDY (SEQ ID NO: 3207) I083D04 26-35 50-66 99-118 DILPDYDFWNPNEDASSLDT (SEQ ID NO: 3133) I083D07 26-35 50-66 99-121 DFQMVRGVFIANPPIYNYYGMDV (SEQ ID NO: 3154) I083D08 26-35 50-66 99-115 DADEGLVEAETTNWFDS (SEQ ID NO: 3126) I083D10 26-37 52-69 102-119  ATKSYDILTRMYYYHMDV (SEQ ID NO: 2748) I083D12 26-35 50-66 99-105 DRTRMDV (SEQ ID NO: 3182) I083E02 26-35 50-66 99-111 VGIKAAAVDNFEY (SEQ ID NO: 2197) I083E03 26-35 50-66 99-108 DEIYNDAFDY (SEQ ID NO: 3105) I083E04 26-35 50-66 99-116 DGDISDSPINNQNYAMDI (SEQ ID NO: 3101) I083E08 26-35 50-66 99-111 RGGTSENYSGMDV (SEQ ID NO: 3209) I083E12 26-35 50-66 99-107 DYPHNAFDI (SEQ ID NO: 3127) I083F02 26-35 50-66 99-118 DVRSDRFWSGGYFHYSGMDV (SEQ ID NO: 3131) I083F04 26-35 50-66 99-110 STLEVGATDFDY (SEQ ID NO: 3199) I083F06 26-35 50-66 99-107 SDDWGAYHI (SEQ ID NO: 3198) I083F08 26-35 50-66 99-111 ERGGRDGDYALDF (SEQ ID NO: 3148) I083F11 26-35 50-66 99-109 ELVGAPGGFDP (SEQ ID NO: 3191) I083G04 26-35 50-66 99-111 VDYTDYEMGAFDL (SEQ ID NO: 3172) I083G05 26-35 50-68 101-110  SVAGRGNFDY (SEQ ID NO: 3208) I083G06 26-35 50-66 99-111 ERGGRDGDYALDF (SEQ ID NO: 3148) I083G08 26-35 50-66 99-114 EGGGDAYDVAPYYFDY (SEQ ID NO: 2204) I083G09 26-35 50-66 99-103 DPFDY (SEQ ID NO: 3134) I083G11 26-35 50-66 99-113 ALLGLPSDFSYYVDV (SEQ ID NO: 3159) I083H04 26-35 50-66 99-114 EGEGDGYNVAPYYFDY (SEQ ID NO: 3160) I083H05 26-35 50-66 99-106 TDYGGFDY (SEQ ID NO: 3092) I083H07 26-35 50-66 99-110 GGVGDSRGVFDP (SEQ ID NO: 3162) I084A03 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084A08 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084B08 26-35 50-66 99-108 ESLTGDAFDI (SEQ ID NO: 3116) I084C02 26-35 50-66 99-109 SPLHFSDAFDI (SEQ ID NO: 3120) I084D03 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084D05 26-35 50-66 99-106 EVGGAFDI (SEQ ID NO: 3157) I084E01 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084E06 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084E10 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084E12 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084F04 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084F07 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084F12 26-35 50-66 99-108 ESLTGDAFDI (SEQ ID NO: 3116) I084G12 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I084H02 26-35 50-66 99-103 DTTDY (SEQ ID NO: 2203) I099B05 26-35 50-66 99-118 GAHYYDRSPSHLKSYWYFDL (SEQ ID NO: 3149) I099G09 26-35 50-66 99-111 VGIKAAAVDNFEY (SEQ ID NO: 2197) I099H01 26-35 50-66 99-111 LGRNYTSSWSLDY (SEQ ID NO: 3181) I099H06 26-35 50-66 99-111 VGIKAAAVDNFEY (SEQ ID NO: 2197) I099H08 26-35 50-66 99-117 GGRYGYYYDGTGYVDAFDI (SEQ ID NO: 3226) I100A01 26-35 50-66 99-109 DNGGGTIGFDY (SEQ ID NO: 2195) I100A10 26-35 50-66 99-113 VRQQIADPPRSFFDP (SEQ ID NO: 3144) I100B03 26-35 50-66 99-109 DNGGGTIGFDY (SEQ ID NO: 2195) I100B04 26-35 50-66 99-109 DNGGGTIGFDY (SEQ ID NO: 2195) I100C03 26-35 50-66 99-113 VRQQIADPPRSFFDP (SEQ ID NO: 3144) BAB2001 31-35 50-65 99-113 EQYDTLTGSPYGMDV BAB2080 31-35 50-66 99-108 LNSLRGGHDY BAB2015 31-35 50-65 99-113 GASSGWYDYYYYMDV BAB2019 31-35 50-66 99-114 DSYDILTDYYNMIMDV BAB2087 31-35 50-66 99-117 GFTGYDILTDYYSVDYFDS BAB2016 31-35 50-66 99-114 DPRYDILTGYLYGMDV BAB2034 31-37 52-69 102-121  EGAHYDILTGHNYYHYGMDV BAB2065 31-35 50-66 99-114 ATYDPLTGYSFDGFD 

1. A method of reducing immunoglobulin levels in a mammal comprising administering a BLyS antagonist and an immunosuppressive drug.
 2. The method of claim 1, wherein the BLyS antagonist is a Fc-fusion protein.
 3. The method of claim 2, wherein the Fc-fusion protein is selected from the group consisting of a TACI-Fc-fusion protein comprising the extracellular domain of TACI or a functional fragment thereof, a BAFF-R-Fc-fusion protein comprising the extracellular domain of BAFF-R or a functional fragment thereof, and a BCMA-Fc-fusion protein comprising the extracellular domain of BCMA or a functional fragment thereof.
 4. The method of claim 1, wherein the BLyS antagonist is a BLyS antibody.
 5. The method of claim 4, wherein the BLyS antibody binds BLyS within a region comprising amino acids 162-275 of SEQ ID NO:
 3228. 6. The method of claim 4, wherein the BLyS antibody is LymphoStat-B.
 7. The method of claim 1, wherein the BLyS antagonist is a TACI antibody.
 8. The method of claim 1, wherein the TACI antibody binds TACI within a region comprising amino acids selected from the group consisting of 72-109 of Genbank Accession No. AAC51790 and 82-222 of Genbank Accession No. AAC51790.
 9. The method of claim 8, wherein the TACI antibody binds both TACI and BCMA.
 10. The method of claim 1, wherein the immunosuppressive drug is selected from the group consisting of cyclophosphamide (CYC), azathioprine (AZA), cyclosporine A (CSA), and mycophenolate mofetil (MMF).
 11. The method of claim 10, wherein the immunosuppressive drug is mycophenolate mofetil (MMF).
 12. The method of claim 1, wherein the BLyS antagonist and the immunosuppressive drug act synergistically to reduce immunoglobulin levels.
 13. The method of claim 1, wherein the immunoglobulin level that is reduced is selected from the group consisting of IgM, IgG, IgA, IgD and IgE.
 14. A method of alleviating a B-cell regulated autoimmune disorder comprising administering to a patient suffering from the disorder a therapeutically effective amount of a BLyS antagonist and an immunosuppressive drug.
 15. The method of claim 14, wherein the BLyS antagonist is a Fc-fusion protein.
 16. The method of claim 15, wherein the Fc-fusion protein is selected from the group consisting of a TACI-Fc-fusion protein comprising the extracellular domain of TACI or a functional fragment thereof, a BAFF-R-Fc-fusion protein comprising the extracellular domain of BAFF-R or a functional fragment thereof, and a BCMA-Fc-fusion protein comprising the extracellular domain of BCMA or a functional fragment thereof.
 17. The method of claim 14, wherein the BLyS antagonist is a BLyS antibody.
 18. The method of claim 14, wherein the BLyS antibody binds BLyS within a region comprising amino acids 162-275 of SEQ ID NO:
 3228. 19. The method of claim 14, wherein the BLyS antibody is LymphoStat-B.
 20. The method of claim 14, wherein the BLyS antagonist is a TACI antibody.
 21. The method of claim 14, wherein the TACI antibody binds TACI within a region comprising amino acids selected from the group consisting of 72-109 of Genbank Accession No. AAC51790 and 82-222 of Genbank Accession No. AAC51790.
 22. The method of claim 20, wherein the TACI antibody binds both TACI and BCMA.
 23. The method of claim 14, wherein the immunosuppressive drug is selected from the group consisting of cyclophosphamide (CYC), azathioprine (AZA), cyclosporine A (CSA), and mycophenolate mofetil (MMF).
 24. The method of claim 23, wherein the immunosuppressive drug is mycophenolate mofetil (MMF).
 25. The method of claim 14, wherein the BLyS antagonist and the immunosuppressive drug act synergistically to reduce immunoglobulin levels.
 26. The method of claim 14, wherein the immunoglobulin level that is reduced is selected from the group consisting of IgM, IgG, IgA, IgD and IgE.
 27. The method of claim 14, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpuria (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes, mellitus, Reynauld's syndrome, Sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, and autoimmune thyroiditis.
 28. The method of claim 27, wherein the autoimmune disease is lupus nephritis.
 29. The method of claim 14, the BLyS antagonist is administered at a dosage of about 1 to about 2.5 mg/kg and the immunosuppressive drug is administered at a dosage of about 1 to about 4 mg/kg.
 30. The method of claim 14, wherein the BLyS antagonist and the immunosuppressive drug is administered in conjunction with therapy using a second immunosuppressive drug selected from the group consisting of nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoid, prednisone, and disease-modifying antirheumatic drugs (DMARDs).
 31. A composition comprising a BLyS antagonist and an immunosuppressive drug.
 32. An article of manufacture comprising a BLyS antagonist and an immunosuppressive drug, and a label wherein the label indicates that the composition is for treating a B cell regulated autoimmune disorder.
 33. The method of claim 1, wherein said BLyS antagonist is also an APRIL antagonist.
 34. The method of claim 14, wherein said BLyS antagonist is also an APRIL antagonist.
 35. The composition of claim 30, wherein said BLyS antagonist is also an APRIL antagonist.
 36. The article of manufacture of claim 31, wherein said BLyS antagonist is also an APRIL antagonist.
 37. A method of reducing immunoglobulin levels in a mammal comprising administering an APRIL antagonist and an immunosuppressive drug.
 38. A method of alleviating a B-cell regulated autoimmune disorder comprising administering to a patient suffering from the disorder a therapeutically effective amount of an APRIL antagonist and an immunosuppressive drug.
 39. A composition comprising an APRIL antagonist and an immunosuppressive drug.
 40. An article of manufacture comprising an APRIL antagonist and an immunosuppressive drug, and a label wherein the label indicates that the composition is for treating a B cell regulated autoimmune disorder.
 41. A method of reducing an immunoglobulin level in a mammal comprising administering a BLyS antagonist and an immunosuppressive drug.
 42. The method of claim 41, wherein the immunoglobulin level is reduced by reducing immunoglobulin production.
 43. The method of claim 41, wherein the BLyS antagonist is a Fc-fusion protein.
 44. The method of claim 43, wherein the Fc-fusion protein is selected from the group consisting of a TACI-Fc-fusion protein comprising the extracellular domain of TACI or a functional fragment thereof, a BAFF-R-Fc-fusion protein comprising the extracellular domain of BAFF-R or a functional fragment thereof, and a BCMA-Fc-fusion protein comprising the extracellular domain of BCMA or a functional fragment thereof.
 45. The method of claim 41, wherein the BLyS antagonist is a BLyS antibody.
 46. The method of claim 45, wherein the BLyS antibody binds BLyS within a region comprising amino acid residues 162-285 of SEQ ID NO:
 3228. 47. The method of claim 45, wherein the BLyS antibody comprises amino acid residues 1-123 of SEQ ID NO:327 and amino acid residues 141-249 of SEQ ID NO:327.
 48. The method of claim 41, wherein the immunosuppressive drug is selected from the group consisting of cyclophosphamide, azathioprine, cyclosporine, and mycophenolate mofetil.
 49. The method of claim 48, wherein the immunosuppressive drug is mycophenolate mofetil.
 50. The method of claim 41, wherein the immunoglobulin level that is reduced is selected from the group consisting of IgM, IgG, IgA and IgE.
 51. A method of treating or ameliorating an autoimmune disorder comprising administering to a patient suffering from the disorder a therapeutically effective amount of a BLyS antagonist and an immunosuppressive drug.
 52. The method of claim 51, wherein the BLyS antagonist is a Fc-fusion protein.
 53. The method of claim 52, wherein the Fc-fusion protein is selected from the group consisting of a TACI-Fc-fusion protein comprising the extracellular domain of TACI or a functional fragment thereof, a BAFF-R-Fc-fusion protein comprising the extracellular domain of BAFF-R or a functional fragment thereof, and a BCMA-Fc-fusion protein comprising the extracellular domain of BCMA or a functional fragment thereof.
 54. The method of claim 51, wherein the BLyS antagonist is a BLyS antibody.
 55. The method of claim 54, wherein the BLyS antibody binds BLyS within a region comprising amino acid residues 162-285 of SEQ ID NO:
 3228. 56. The method of claim 54, wherein the BLyS antibody comprises amino acid residues 1-123 of SEQ ID NO:327 and amino acid residues 141-249 of SEQ ID NO:327.
 57. The method of claim 51, wherein the immunosuppressive drug is selected from the group consisting of cyclophosphamide, azathioprine, cyclosporine, and mycophenolate mofetil.
 58. The method of claim 57, wherein the immunosuppressive drug is mycophenolate mofetil.
 59. The method of claim 51, wherein the BLyS antagonist and the immunosuppressive drug reduce an immunoglobulin level.
 60. The method of claim 59, wherein the immunoglobulin level is reduced by reducing immunoglobulin production.
 61. The method of claim 59, wherein the immunoglobulin level that is reduced is selected from the group consisting of IgM, IgG, IgA and IgE.
 62. The method of claim 51, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, nephritis associated with systemic lupus erythematosus, inflammatory bowel disease, idiopathic thrombocytopenia purpura, autoimmune thrombocytopenic purpura, Multiple Sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, Myasthenia Gravis, vasculitis, diabetes mellitus, Raynaud phenomenon, Sjögren's syndrome, glomerulonephritis, autoimmune hepatitis, and autoimmune thyroiditis.
 63. The method of claim 62, wherein the autoimmune disease is nephritis associated with systemic lupus erythematosus.
 64. The method of claim 51, wherein the BLyS antagonist is administered at a dosage of 1 mg/kg to 10 mg/kg of the patient's body weight.
 65. The method of claim 51, wherein the method further comprises administering a drug selected from the group consisting of nonsteroidal anti-inflammatories, glucocorticoid, prednisone, methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial drug, cyclophosphamide, chlorambucil, gold, etanercept, infliximab, leflunomide, an anti-TNF antibody, LJP 394, and prednisolone.
 66. A composition comprising a BLyS antagonist and an immunosuppressive drug.
 67. An article of manufacture comprising a BLyS antagonist and an immunosuppressive drug, and a label wherein the label indicates that the composition is for treating an autoimmune disorder.
 68. The method of claim 41, wherein the BLyS antagonist is also an APRIL antagonist.
 69. The method of claim 51, wherein the BLyS antagonist is also an APRIL antagonist.
 70. The composition of claim 66, wherein the BLyS antagonist is also an APRIL antagonist.
 71. The article of manufacture of claim 67, wherein the BLyS antagonist is also an APRIL antagonist. 